Methods, substrates, and systems useful for cell seeding of medical grafts

ABSTRACT

Described are methods, cell growth substrates, and devices that are useful in preparing cell-containing graft materials for administration to patients. Tubular passages can be defined in cell growth substrates to promote distribution of cells into the substrates. Also described are methods and devices for preparing cell-seeded graft compositions, methods and devices for preconditioning cell growth substrates prior to application of cells, and cell seeded grafts having novel substrates, and uses thereof.

REFERENCE TO RELATED APPLICATION

This application is a divisional of U.S. patent application Ser. No.13/115,347, filed May 25, 2011, which claims the benefit of U.S.Provisional Patent Application Ser. No. 61/348,135 filed May 25, 2010entitled METHODS, SUBSTRATES, AND SYSTEMS USEFUL FOR CELL SEEDING OFMEDICAL GRAFTS, each of which is hereby incorporated by reference in itsentirety.

BACKGROUND

The present invention relates generally to medical materials andprocedures, and in specific aspects relates to cell-containing medicalgrafts and methods, substrates and devices useful for preparing them.

The field of tissue engineering has demonstrated significant promise toimprove medical treatments for patients across a broad variety ofconditions or injuries. One area of study has been that of implantablegraft materials that contain viable cells from the patient or from othersources. With regard to the harvest and re-introduction of cells fromthe patient, termed autologous cellular treatment, methods and systemsare known for treating a tissue sample from the patient to result in acellular preparation that can be re-introduced to the patient. It hasbeen proposed that such methods and systems can be used “bedside” in ahospital setting, e.g. in a single hospital procedure or visit in whichthe biological tissue sample is obtained from the patient, processed toa cellular composition, and re-introduced into the patient.

In certain modes of use, cells to be introduced into the patient can becombined with a cell growth substrate to form a cell-containingimplantable graft. Sometimes, these uses involve a culture period inwhich the number of cells is expanded after application to the cellgrowth substrate. Other modes of use do not involve such expansion.Rather, the cells are applied to the cell growth substrate and implantedwithout a culture period.

Despite demonstrated promise, the clinical implementation ofcell-containing graft materials has been slow. Needs exists for moreconvenient and/or effective ways or materials for combining cells withcell growth substrates so that they are situated for survival and oftenexpansion in the patient. In certain of its aspects, the presentinvention is addressed to these needs.

SUMMARY

In some of its embodiments, the present invention provides methods, cellgrowth substrates, and devices that are useful in preparingcell-containing graft materials for administration to patients. Cellgrowth substrates of the invention can include features enabling theirenhanced combination with cell suspensions. In certain embodiments, suchfeatures include tubular passages defined in the cell growth substratesto promote distribution of cells during application of cell-suspensionsto the substrates. Additional embodiments herein disclosed relate tomethods and devices for preparing cell-seeded, flowable matrix graftcompositions; to methods and devices for pre-conditioning cell growthsubstrates prior to application of cell compositions to them; toautomated methods and devices for preparing cell-seeded implantablegrafts by combining cellular compositions and substrate materials, forexample including features for distributing the cells throughout avolume of the substrate materials; and to particulate forms of cellgrowth substrates and their use to prepare flowable cellular graftmaterials.

In one particular embodiment, provided is a cell growth substrate forsupporting the growth of cells. The substrate includes at least oneelongate tubular passage having passage walls defining a lumen extendingfrom a first lumen opening at a surface of the cell growth substratebody into an interior region of the cell growth substrate. The lumen canbe configured to receive flow of a cell-containing liquid medium todistribute cells into the interior region of the cell growth substrate.The cell growth substrate can be a cell growth matrix. The substrate cancomprise collagen and/or can also comprise a synthetic polymeric tubularelement exterior of the substrate and fluidly coupled to the lumenopening. The substrate can include a plurality of the tubular passages.The at least one tubular passage can include at least one primarytubular passage and at least one secondary tubular passage branchingfrom the primary tubular passage. The substrate can comprise aremodelable collagenous extracellular matrix sheet material, and thesheet material can retain growth factors, glycosaminoglycans, and/orproteoglycans from an animal source tissue for the sheet material.

In another embodiment, provided is a method for preparing a cell-seededmaterial that includes connecting a cell growth substrate, such as onedescribed in the paragraph above, to a source of a liquid suspension ofcells, wherein the connecting includes fluidly associating the elongatepassage with a transport lumen of the source. The method also includestransporting amounts of the liquid suspension of cells through thetransport lumen and into the elongate passage so as to deliver cells tothe interior region of the substrate. The connecting step can includeinserting the substrate into a cell-seeding chamber having an input tubefluidly connected to the source of liquid suspension of cells, andfluidly coupling the input tube to the first lumen opening of thesubstrate.

In a further embodiment, provided is a method for preparing acell-seeded composition for delivery to a patient. The method includescombining a fluid extracellular matrix particulate composition,comprising particles of extracellular matrix and a liquid medium, with aliquid cell suspension to form a cellular fluid composition. The methodcan also include mixing the cellular fluid composition and/or causingthe cellular fluid composition to gel. The gel-causation can include astep of altering the pH of the cellular fluid composition and/oraltering the temperature of the cellular fluid composition.

A further embodiment provides a method for preparing and administering acellular graft. The method includes applying a serum protein compositionto a biologically compatible substrate suitable for administration to apatient, to prepare a preconditioned substrate including the serumprotein composition and the biologically compatible substrate. Cells areadded to the preconditioned substrate to prepare a cellular graft, andthe cellular graft is administered to a patient. The applying a serumprotein composition step can include spray applying the composition. Thestep of adding cells can include spraying a liquid cell suspension ontothe preconditioned substrate and/or passing a liquid cell suspensioninto a tubular passage of the substrate.

In another embodiment, provided is a device for seeding a matrix withcells. The device includes a first chamber for receiving a liquidsuspension of cells, and a second chamber for receiving a cell growthmatrix material to be seeded with the cells. The seeding device furtherincludes a passageway for transfer of amounts of the liquid suspensionof cells from the first chamber to the second chamber, a device fordetecting at least one condition of the liquid suspension of cells, andan application device for applying the amounts of the liquid suspensionof cells to a matrix material received in the second chamber. Theapplication device can include at least one spray nozzle or at least onecannula having a lumen. The seeding device can also include a mechanismfor moving the spray nozzle or cannula. Where the application deviceincludes a cannula, the moving mechanism can be operable to withdraw thecannula proximally while dispensing a liquid suspension of cells from anopening of the cannula such as a distal-most opening. The seeding devicecan also include a registration structure for holding the matrixmaterial in a predetermined position relative to the application device.The seeding device can also include a distribution assist deviceassociated with the second chamber and operable to facilitatedistribution of cells within the matrix material after application ofthe cells to the matrix material by the application device. Thedistribution assist device can be operable to generate a magnetic fieldacross the matrix material; can be a mixer operable to generate flow ina liquid received in the second chamber; can be operable to generate apressure gradient within the second chamber; can be operable to move acell growth substrate received in the second chamber; and/or can beoperable to rotate the second chamber to distribute cells within thecell growth matrix material at least partially by centrifugal force.When present, the mixer operable to generate flow can be operable togenerate pulsatile, bi-directional flow in a liquid received in thesecond chamber.

A further embodiment provides a device for preparing a flowable,cell-seeded graft. The device includes a first chamber containing aliquid suspension of cells, and a second chamber containing a flowablecell growth substrate material to be seeded with the cells, the secondchamber fluidly connected to the first chamber so that the liquidsuspension of cells can be combined with the flowable cell growthsubstrate material to form a flowable, cell-seeded graft material. Thedevice also includes a mixer operable to mix the flowable, cell-seededgraft material. The mixer can be a static mixer positioned within a flowpath for the flowable, cell-seeded graft material, or a rotary mixer.The first and/or second chamber can be a passage.

In another embodiment, provided is a method for preparing a cell-seededsubstrate for treating a patient, including a human patient, andpotentially also for treating the patient. The method includesprocessing a tissue sample of the patient to obtain a suspension ofcells of the patient, and loading a cell growth substrate into anincubation chamber of a cell seeder device. The method also includesinitiating operation of the cell seeder device, wherein the operationcauses detection of at least one condition of the suspension of cells,and combination of the cells of the suspension with the cell growthsubstrate to form a cell-seeded substrate. In a method for treating thepatient, the method also includes administering the cell-seededsubstrate to the patient.

Also provided is a method for preparing a cell seeded matrix thatincludes admixing cells with a gellable composition having a firstviscosity to form a gellable cell mixture, and applying the gellablecell mixture to a cell growth substrate. The method also includescausing the gellable cell mixture to gel to form a cellular gel incontact with said cell growth substrate. The gel can have a secondviscosity which is greater than the first viscosity. The gelling of themixture can be caused by any suitable measure or combination thereof,including for example altering (e.g. raising or lowering) the pH of themixture and/or altering (e.g. raising or lowering) the temperature ofthe mixture. The mixture can be contacted with the substrate so as toprovide a layer on an outer surface of the substrate, and/or can bedistributed substantially homogenously through the substrate (e.g. wherethe substrate is porous), or at least a portion of the substrate.Subsequent gelling of the mixture can provide an external cellularizedgel layer and/or a cellularized gel distributed substantiallyhomogeneously through all or a portion of the substrate.

In a further embodiment, provided is a cell-seeded graft that includesan extracellular matrix substrate material, a collagenous gel applied tothe substrate material, and cells adhered to the substrate material, tothe gel, or to both. The collagenous gel can be comprised of anextracellular matrix hydrolysate comprising native collagen and at leastone additional native bioactive substance from a source tissue for theextracellular matrix hydrolysate. The at least one additional nativebioactive substance can include growth factor(s), glycosaminoglycan(s)and/or proteoglycan(s).

A further embodiment provides a method for preparing a cellular graftfor treating a patient, including a human patient, and also potentiallyfor treating the patient. The method includes obtaining serum from thepatient, and processing a tissue sample of the patient to obtain apopulation of cells of the patient. The method further includes applyingthe serum to a matrix substrate to prepare a serum-preconditioned matrixsubstrate, and applying the population of cells to the preconditionedmatrix substrate to form a cellular graft. In a method for treating thepatient, the method can also include administering the cellular graft tothe patient.

In a still further embodiment, provided is a method for preparing amaterial for treating a patient, and potentially also for treating thepatient. The method includes combining a suspension of cells with aparticulate cell growth substrate, and incubating the suspension ofcells in contact with the particulate cell growth substrate to formcellularized particulate bodies having cells attached to particles ofthe particulate cell growth substrate, wherein the incubating is for aduration and under conditions such that significant expansion of thenumber of cells does not occur. In a method for treating the patient,the method can also include administering the cellularized particulatebodies to the patient. In certain forms, the incubating of the methodcan be for a duration and under conditions effective to achieveattachment of at least 20% of the cells in the suspension to theparticles but without expansion of the number of cells.

Another embodiment provides a cellular graft for treating a patient, andpotentially also use of the cellular graft in a method for treating apatient with the cellular graft, and/or in a method for manufacturing amaterial for the treatment of a patient. The cellular graft includescellularized particulate bodies comprised of cells attached toextracellular matrix particles. For use in treatment, the cellular graftcan be introduced into the patient.

Another embodiment provides a cellular graft material including aparticulate cell growth substrate material and cells attached toparticles of the cell growth substrate material. The cells compriseendothelial progenitor cells, muscle derived cells, or a combinationthereof. The particles can be particles of a naturally-derivedextracellular matrix sheet material isolated from an animal source andoptionally processed so as to retain endogenous growth factor(s),glycosaminoglycan(s) and/or proteoglycan(s). Such particles of anaturally-derived extracellular matrix sheet material can be prepared bycomminuting the sheet material to form randomly generated particles,and/or by cutting the sheet material to form particles of regular shape(e.g. in the form of circular, ovoid and/or polygonal shapes).

A further embodiment provides a cellular graft that comprises a filamentcomprised of an extracellular matrix material, and a population of cellsattached to the filament. The filament can be a segment of anaturally-derived extracellular matrix sheet material isolated from ananimal source and processed so as to retain endogenous growth factor(s),glycosaminoglycan(s) and/or proteoglycan(s).

Another embodiment provides a cellular graft that includes a cell growthsubstrate in the form of a filament, and a population of cells attachedto the filament, where the cells include endothelial progenitor cells.This cellular graft can be introduced into tissue of a patient in afurther embodiment of the invention which provides a method forvascularizing tissue of a patient.

Another embodiment provides a method for preparing a cellular graft. Themethod includes providing a first cell growth substrate sheet, and firstapplying a cellular composition to a surface of the first cell growthsubstrate sheet to form a first cell-seeded surface. After said firstapplying, the method includes stacking a second cell growth substratesheet against said first cell-seeded surface. The method can alsoinclude second applying a cellular composition to a surface of thesecond cell growth substrate sheet.

Another embodiment provides a cellular graft that includes a first cellgrowth substrate sheet and a second cell growth substrate sheet stackedon the first sheet. The graft also includes a layer of seeded cellsbetween the first sheet and the second sheet.

Another embodiment provides a device for preparing a cell-seeded graft.The devices includes an incubation chamber for receiving a cell growthsubstrate and an application device for applying a liquid cellsuspension to the cell growth substrate, the application devicecomprising a plurality of cannulae for dispensing amounts of thesuspension. The device can be configured to retract the plurality ofcannulae while dispensing amounts of the suspension therefrom.

In still another embodiment, provided is a cell growth substratecomposition that includes a particulate material comprising sheet-formcell growth substrate particles have a compact shape. In some forms, atleast 25% of the sheet-form substrate particles, when considered in theplane of the sheet, have a first, maximum cross sectional dimension axiswhich is no more than about two times the length of a second crosssectional axis taken on a line perpendicular to and centered upon themaximum cross sectional dimension axis. The substrate particles can havea maximum cross sectional dimension in the range of about 20 microns toabout 2000 microns. The substrate particles can be particles of anaturally-derived extracellular matrix sheet material isolated from ananimal source and optionally processed so as to retain endogenous growthfactor(s), glycosaminoglycan(s) and/or proteoglycan(s). Such particlesof a naturally-derived extracellular matrix sheet material can beprepared for example by cutting the sheet material to form particles ofa compact shape (e.g. in the form of circular, ovoid and/or polygonalshapes).

Another embodiment provides an extracellular matrix composition thatincludes a particulate extracellular matrix material comprisingsheet-form extracellular matrix particles having a compact shape. Atleast 25% of the sheet-form extracellular matrix particles, whenconsidered in the plane of the sheet, can have a first, maximum crosssectional dimension axis which is no more than about two times thelength of a second cross sectional axis taken on a line perpendicular toand centered upon the maximum cross sectional dimension axis. Theextracellular matrix particles can have a maximum cross sectionaldimension in the range of about 20 microns to about 2000 microns. Theextracellular matrix particles can be particles of a naturally-derivedextracellular matrix sheet material isolated from an animal source andoptionally processed so as to retain endogenous growth factor(s),glycosaminoglycan(s) and/or proteoglycan(s). Such particles of anaturally-derived extracellular matrix sheet material can be preparedfor example by cutting the sheet material to form particles of a compactshape (e.g. in the form of circular, ovoid and/or polygonal shapes). Theextracellular matrix composition can also include cells attached to theparticles, with the cells in certain embodiments covering substantiallythe entire outer surface of the particles and/or forming a substantiallyconfluent monolayer on the surface. The cells can include endothelialcells, endothelial progenitor cells, or a mixture thereof, and/or can beclonal. In some forms, the cells can consist of endothelial cells,endothelial progenitor cells, or a mixture thereof.

In another embodiment, provided is a cell growth substrate article thatincludes a cell growth substrate material and an encapsulating materialencapsulating the cell growth substrate material and configured todirect flow of a fluid medium through the substrate material. Theencapsulating material can define at least a first opening, and at leasta second opening spaced from the first opening.

Another embodiment provides a cellular graft that includes a mixedpopulation of cells derived from adipose tissue and including stemcells, endothelial progenitor cells, leukocytes, endothelial cells, andvascular smooth muscle cells. The cellular graft also includes a cellgrowth substrate that is comprised of an extracellular matrix materialand/or that is in particulate form. The extracellular matrix materialcan include a retained bioactive component native to a source tissue forthe extracellular matrix material. The retained bioactive component canbe a growth factor(s), glycosaminoglycan(s) and/or proteoglycan(s). Whenused, a particulate cell growth substrate can comprise sheet-formparticles. For these purposes, sheet form particles of anaturally-derived extracellular matrix sheet material can be preparedfor example by cutting the sheet material to form particles, for exampleof a compact shape (e.g. in the form of circular, ovoid and/or polygonalshapes).

In a further embodiment, provided is a cellular graft that includes apopulation of endothelial progenitor cells and a particulate cell growthsubstrate. The particulate cell growth substrate can include sheet-formparticles and/or can include an extracellular matrix material.Extracellular matrix particles for these purposes can be particles of anaturally-derived extracellular matrix sheet material isolated from ananimal source and optionally processed so as to retain endogenous growthfactor(s), glycosaminoglycan(s) and/or proteoglycan(s). Such particlesof a naturally-derived extracellular matrix sheet material can beprepared for example by cutting the sheet material to form particles ofa compact shape (e.g. in the form of circular, ovoid and/or polygonalshapes), or can be fragments of the sheet material, e.g. generated byrandomly comminuting the sheet material.

Another embodiment provides a system for seeding a matrix with cells.The system includes a chamber for combining cells with a cell growthsubstrate to be seeded with the cells. The system also includes amechanism for assessing adherence of the cells to the substrate. Themechanism for assessing can comprise a cell counter. Related methods forseeding a matrix comprise the steps of combining cells with a cellgrowth substrate to be seeded with the cells, and assessing adherence ofthe cells to the substrate. In treatment methods, the methods can alsoinclude administering the substrate, seeded with adhered cells, to apatient, including a human patient.

Additional embodiments of the invention as well as features andadvantages thereof will be apparent from the further descriptionsherein.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 provides a side view of one embodiment of a cell growth substrateof the invention.

FIG. 2 provides a side view of another embodiment of a cell growthsubstrate of the invention.

FIG. 3 provides a top view of the substrate of FIG. 1.

FIG. 4 shows a top view of an alternative cell growth substrate of theinvention.

FIG. 5 shows a top view of a further embodiment of a cell growthsubstrate.

FIG. 6 shows a top view of another embodiment of a cell growthsubstrate, having a manifold feed.

FIG. 7 shows a top view of another embodiment of a cell growthsubstrate.

FIG. 8 provides a top view of another embodiment of a cell growthsubstrate, having larger primary channels and smaller secondary channelswithin the substrate.

FIG. 9 provides a schematic diagram of one embodiment of a system forapplying a cellular composition to a cell growth substrate.

FIG. 9A illustrates one embodiment of certain subcomponents of thesystem of FIG. 9.

FIG. 9B illustrates another embodiment of certain subcomponents of thesystem of FIG. 9.

FIG. 10 provides a schematic view of another embodiment of a device forcombining a cellular composition with a cell growth substrate.

FIG. 11 is a digital image of a compact sheet particle of adecellularized ECM sheet, compositions of which are useful as cellgrowth substrates.

FIG. 11A provides an illustration of a flowable cell graft material ofthe invention, combined with a delivery device.

FIG. 12 provides an illustration of cellularized filament grafts of theinvention.

FIG. 12A provides an illustration of a cellularized filament graft ofthe invention having a distal retention barb.

FIG. 12B provides an illustration of the graft of FIG. 12A received in adelivery needle cannula in use to implant the graft.

FIG. 13 provides an illustration of a cell graft of the invention havingmultiple, stacked cell growth substrate sheets.

FIGS. 14 to 16 provide illustrations of cell growth substrate articlesof manufacture of the invention.

FIGS. 17 and 18 provide illustrations of additional cell graftconstructs of the invention.

DETAILED DESCRIPTION

For the purpose of promoting an understanding of the principles of theinvention, reference will now be made to embodiments, some of which areillustrated in the drawings, and specific language will be used todescribe the same. It will nevertheless be understood that no limitationof the scope of the invention is thereby intended. Any alterations andfurther modifications in the described embodiments, and any furtherapplications of the principles of the invention as described herein arecontemplated as would normally occur to one skilled in the art to whichthe invention relates.

As disclosed above, aspects of the present invention relate to methods,materials and systems for combining cells with substrate materials toprepare compositions that can be used in certain embodiments as medicalimplants for patients.

Cell Growth Substrates Having Defined Internal Passages

Referring now to FIGS. 1-8, shown are various embodiments of cell growthsubstrate constructs that are particularly useful for combination withcellular compositions to form cell-containing graft materials.

In particular, FIG. 1 provides a side view of a matrix substrate 20.Substrate 20 includes a substrate body 21 preferably comprised of aporous matrix material suitable for supporting cellular growth.Substrate body 21 includes internally defined passages 22 extending intoan interior region of the substrate body 21. In the depicted embodiment20, substrate body 21 includes a first sheet 23 connected to a secondsheet 24 in such a fashion as to define passages 22 between sheet 23 andsheet 24. Sheet 23 and sheet 24 are connected to one another along aconnected region 25 in areas surrounding passages 22. Connected region25 may, for example, be a bonded, fused, glued, cross-linked, sutured,or other arrangement establishing a close connection between the opposedfaces of sheet 23 and sheet 24. Tubular passages 22 thus include apassage wall having a portion 26 defined by a face of sheet 23 and aportion 27 defined by an opposed face of sheet 24. In the illustratedembodiment, regions 26 and 27 are generally rounded, providing asubstantially circular or ovate cross section to passages 22. It will beunderstood that other arrangements are also suitable, including passageshaving polygonal or irregular cross sectional shapes. Connected region25 has terminus regions 28 and 29 at the lumens of passages 22. Terminusregions 28 and 29 can thus occur as seams along the walls of passages22.

To prepare substrate 20, a passage-forming element or elements can beplaced between sheet 23 and sheet 24, and these sheets can then beconnected to one another in connection regions 25 thus sandwiching thepassage-forming element or elements between the sheets. As noted above,this connection can be by bonding, fusing, gluing, or otherwise. Wherean opening of passage 22 to the outermost edge of substrate 20 isdesired, the passage-forming element or elements can extend to or beyondthe outermost edges of sheets 23 and 24 as they are layered against oneanother, thus providing such openings in the finished product. In suchpreparative methods, the passage-forming element or elements can berods, bars, comb structures, wires, tubes, or any other element suitablefor maintaining space between the sheets 23 and 24 for providing apassage in the completed product. In embodiments in which thepassage-forming element or elements will be removed after formation ofthe connected regions 25, it is desired that the passage-formingelement(s) have an exterior surface that will sufficiently avoidsticking or bonding to the opposed faces of sheets 23 and 24 such thatthe passage-forming element(s) can be removed after formation of theconnected regions 25 to leave passages 22 intact.

In certain embodiments, the passage-forming element(s), or at leastportions thereof, will be left resident within the finished product.Referring now to FIG. 2, one such embodiment is shown. Substrate 30includes the same features as matrix 20 and as a general matter can beconstructed in the same fashion. Thus, substrate 30 includes a body 31,passages 32 to interior regions of the body 31, and sheets 33 and 34connected to one another. Additionally, substrate 30 includes residenttubular elements 35 received between sheets 33 and 34. Tubular elements35 can be made from biocompatible polymers or other suitable materialsand can remain in substrate 30 for implantation into a patient. Tubularelements 35 can comprise persistent polymers (non-bioresorbable) orbioresorbable polymers. In certain embodiments, tubular elements 35comprise bioresorbable polymers and can be completely resorbed afterimplantation in a patient. In the manufacture of substrate 30, tubularelements 35 can be placed between sheets 33 and 34, and then the sheetscan be connected as discussed above in connection with substrate 20(FIG. 1). Alternatively, a device such as substrate 20 can first beprepared, and tubular elements can thereafter be inserted into thepassages 22. Tubular elements 35 can include porous walls, or holes orperforations in their walls, to allow the transmission of a flowablecell suspension through the walls of tubular elements 35 and intosurrounding regions of the graft body 31. In certain forms, the tubularelements 35 are constructed of material that is less absorptive to anaqueous cellular suspension than the material of the body 31, and/orremains more rigid than the material of body 31 when wet so as to retainan open passage, and thus can serve to better transmit a flow of anaqueous cellular suspension along passages 32 to reach interior regionsof the substrate 31. The porosity of or perforations in tubular elements35 can be controlled to optimize wetting and cell seeding of thesubstrate 31 upon inputting a flow of an aqueous cellular suspensioninto passages 32.

With reference now to FIG. 3, shown is a top view of the substratedevice 20 of FIG. 1. Passages 22 are shown in phantom (dotted lines).Substrate 20 as depicted is a generally rectangular structure includinga first pair of generally parallel sides 40 and 41, and a second pair ofgenerally parallel sides 42 and 43 perpendicular thereto. Passages 22have an opening exposed at side 40 of substrate 20. Passages 22 in thedepicted embodiment are blind holes, and have a terminus 44 within theinterior of body 21, spaced a distance “d” from side 41 of body 21. Inthis fashion, fluid compositions such as aqueous cellular suspensionscan be passed into passages 22 from side 40, and will not be carried allthe way to the opposite side 41 by the passages 22. This can provide anenhanced ability to generate fluid pressure within passages 22 to morerapidly disperse a cell suspension and/or another fluid, such as asubstrate preconditioning medium, into adjacent regions of body 21. Incertain modes of use, the blind hole passages 22 can be subjected tofluid pressure by fluid introduced through the openings to passages 22at side 40, to drive the fluid and dissolved or suspended (e.g. cells)materials into and through the volume of body 21.

With reference to FIGS. 4-8, shown are alternative embodiments ofsubstrate devices defining internal passages. Specifically withreference to FIG. 4, shown is a cell growth substrate 50 having featuressimilar to those of substrate 20 of FIGS. 1 and 3, except havingpassages that extend completely from a first perimeter to a secondperimeter of the substrate. In particular, substrate 50 includes a body51 of generally rectangular shape having a first side 52, a second side53 generally parallel with side 52, a third side 54, and a fourth side55 generally parallel with side 54. Substrate 50 includes a plurality ofpassages 22 extending through the material of body 51. The passages 22have a first group of openings 56 occurring on side 52 of substrate 50,and second group of openings 57 occurring on side 53 of substrate 50opposite to side 52. In this fashion, passages 22 present openings 56and 57 at spaced locations on substrate body 51 and in the illustratedembodiment on opposed sides 52 and 53. A flowable composition includingcells can be passed through passages 22 from openings 56 to openings 57to allow the cells to penetrate, contact and adhere to the material ofbody 51, so as to populate the substrate 50 with cells.

With reference to FIG. 5 shown is a cell growth substrate 60 similar tosubstrate 50 of FIG. 4, except having first and second sets of passageswhich are transverse to one another. Thus, substrate 60 includes asubstrate body 61 having a first side 62, a second side 63, a third side64, and a fourth side 65. Substrate 60 includes a first plurality ofpassages 22 having openings 66 at side 62 and opposed openings 67 atside 63 of body 61. Substrate 60 includes a second plurality of passages68 which pass through the body 61 in a direction transverse to that ofpassages 22, and in the illustrated embodiment substantiallyperpendicularly to the direction of passages 22. Passages 68 includeopenings 69 occurring in side 64, and opposed openings 70 occurring inside 65. In certain embodiments, passages 68 are fluidly coupled to andintersect passages 22 at passage intersections 71 distributed throughoutbody 61. In use, substrate 60 can be populated with cells by flowingcellular compositions through passages 22 and through passages 68. Inthis regard, fluid cellular compositions can be passed through passages22 and 68 simultaneously, or at different times, or using a combinationof these during a cell populating operation. In embodiments in whichpassages 22 and 68 intersect one another and are thus fluidly coupled,the simultaneous passage of cellular compositions or other fluidssimultaneously through passages 22 and 68 can create fluid flowconditions at intersections 71, such as turbulence, eddies, or at leasta partial redirection of net flow along a vector that is aligned withneither passages 22 nor 68, so as to facilitate driving the fluid out ofthe passages and into the surrounding volumes of material making upsubstrate body 61. This can provide a more rapid or efficientdistribution of cells or other substances throughout the volume ofsubstrate body 61.

Referring to FIG. 6, shown is a cell growth substrate 80 having aplurality of passages 22 fed by a common manifold structure.Specifically, substrate 80 includes a body 81 having a plurality ofpassages 22 extending therethrough. A common feed passage 82 fluidlycouples to a plurality of passages 22. Common feed passage 82 is in turnfluidly coupled to input passage 83 having an opening 84 to the exteriorof body 81. Passages 22 each have an opening 85 to the exterior of body81. In use, a fluid such as a cellular composition can be passed intoinput passage 83 through opening 84, whereupon the fluid compositionpasses into common passage 82 which in turn distributes the fluidcomposition into and through passages 22, exiting via openings 85. Inthis manner, a fluid cellular or other composition can be circulated andpotentially recirculated through substrate 80 to populate the substratewith cells. As with the other embodiments described herein, toaccomplish this, passages 22 and potentially also passages 82 and 83 canbe permeable to the composition to allow escape into the adjacentvolumes of body 81.

Referring now to FIG. 7, shown is another cell growth substrate 90.Substrate 90 includes a body 91 and first, second, third, and fourthsides 92, 93, 94, and 95, respectively. Body 91 includes a tortuouspassage 96 extending through the material of body 91 and having a firstexternal opening 97 and a second external opening 98 spaced therefrom.In the illustrated embodiment, tortuous internal passage 96 includes afirst plurality of bends 99 occurring opposite a second plurality ofbends 100, with the bends 99 and 100 situated in opposed directions.Generally straight passage segments 101 interconnect bends 99 and 100.In this fashion, tortuous passage 96 takes on a generally repeatingsinusoidal wave shape as it traverses from opening 97 to opening 98.Cell-containing fluid can be circulated from opening 97 through thetortuous passage 96 and to opening 98, during which cells and fluidescape passage 96 to provide cell seeding through the volume ofsubstrate 91.

Referring now to FIG. 8, shown is a cell growth substrate 110 sharingmany features in common with substrate 90 discussed above, which arecorrespondingly numbered. Substrate 110, however, also includes aplurality of secondary passages 122 fluidly coupled to and leading fromprimary passage 116, with the secondary passages having diameters orcross sectional areas smaller than tortuous passage 116. Secondarypassages 122 can thus receive flow of cellular fluid passed throughprimary passage 116 and help to distribute the fluid and accompanyingcells throughout the substrate body 111.

It will be understood that in additional embodiments of the cell growthsubstrates depicted in FIGS. 1 and 3-8, separate tubular elements can bereceived within portions of or all of the defined internal passages, asdiscussed above in conjunction with FIG. 2. Additionally, in preferredembodiments, sheets (e.g. 23 and 24) used to prepare the illustratedsubstrates can be comprised of decellularized ECM tissue sheets,desirably retaining native (endogenous) bioactive component(s), asdescribed hereinafter. As well, the bodies of the substrates illustratedin FIGS. 1-8 can be comprised or constituted of a porous sponge or foammatrix, which can be formed for example by casting a matrix-formingmaterial, e.g. any of those described herein, around the passage andthen be removed or left resident in the finished product to contributeto the passage, as discussed above.

Cell/Substrate Processing Systems and Methods

In additional aspects of the invention, provided are devices for theautomated seeding of substrates with cells. FIG. 9 provides a schematicdiagram of one embodiment of an automated cell seeding system 200.System 200 can optionally include a raw tissue processing module 201that processes raw or early-stage tissue samples to provide an output ofa single cell suspension derived from the tissue samples. Processingmodule 201 can thus operate to perform one or more functions common tothis purpose, including for example physical disruption of tissue (e.g.maceration), enzymatic (e.g. collagenase, trypsin) or other chemicaldisassociation of tissue, and/or washing of tissue or cellularcomponents with saline or other suitable wash media. Tissue processingmodule 201 can also perform cell separation or concentration stepsincluding for example immunochemical selection of specific types orgroups of cells, fractionation, filtration, sedimentation, or othersimilar operations. Module 201 may also include features to expand cellpopulations, for example providing growth substrates or growth media andsuitable incubation conditions for expanding cell number. Illustrativesystems for tissue processing to output cell suspensions are disclosedin US Patent Publication No. US2005/0025755, published Feb. 3, 2005,International Publication No. WO2007/009036, published Jan. 18, 2007, USPatent Application Publication No. US2008/0014181, published Jan. 17,2008, and US Patent Application Publication No. US2006/0141623,published Jul. 29, 2006. These publications are each hereby incorporatedherein by reference in their entirety, for their teaching of tissueprocessing methods and equipment for achieving single cell suspensionssuitable for use in module 201 of system 200.

The cell suspension output of module 201 is transferred via conduit 202to cell suspension chamber 203. A device 204 for detecting at least onecondition of the cell suspension is provided, to provide input forpotential adjustment of the cell suspension prior to application to asubstrate and/or for optimizing parameters for application of thesuspension to a substrate. For example, device 204 may be operative todetermine the concentration of cells in the cell suspension in chamber203 and is operably associated with chamber 203. By way of non-limitingexample, device 204 can be a cell counting device such as a Coultercounter, which arrives at a cell count value based upon electricalresistance changes of a liquid filled channel during passage of thecells through the channel. This resistance change can be sensed aselectric current or voltage pulses, which can be correlated to theconcentration of the cells within the cell suspension. The device 204may also be a device that utilizes light scattering, such as forwardlight scattering using a laser, to calculate a cell concentration value.The device 204 may also be a device that uses flow cytometry todetermine the cell concentration within the cell suspension. Other meansfor measuring cell concentration will be apparent to those skilled inthe art, and it will be appreciated that the particular means formeasuring cell concentration is not critical to the presently disclosedsystems and methods.

To facilitate the cell concentration analysis, device 204 can draw off asample of the cell suspension via conduit 205, for analysis. Optionally,device 204 can return the cell sample to the chamber 203 after theanalysis via conduit 206, unless this would cause unwanted contaminationof the cell suspension within chamber 203, in which case the drawnsample can be discarded via a waste line (not shown). Device 204 and/orthe chamber 203 may include appropriate valves and optional pumps (notshown) for the movement of cell samples through the conduits 205, 206 aswill be apparent to those skilled in the art. Such valves and/or pumpsmay be under the control of process controller 229 as discussed ingreater detail hereinbelow.

System 200 can also include a processing media unit 207 fluidly coupledto chamber 203 via conduit 208. As with all other conduits in whichfluid media are to be transferred in system 200, conduit 208 can beassociated with optional valves (not shown) and a pump 209 operable topower the transfer of fluid from media unit 207 to chamber 203. It willbe understood that pump 209 can be provided by a shared pump duty inwhich a single pump powers fluid transfer amongst some or all of thevarious chambers or other components of system 200. Thus, althoughseveral separate pumps are discussed in conjunction with system 200, itwill be understood that these pump operations could be shared orseparate, or combinations thereof, within the operation of system 200.Such valves and/or pump may be under the control of process controller229 as discussed in greater detail hereinbelow. Processing media unit207 can include one or more chambers containing, or into which can becharged, any of a variety of media for treating or processing the cellsuspension composition output from the module 201. These media couldinclude, for example, wash media, cell culture media, or other materialsfor readying the cells for application to the cell growth substrate,including for example differing media for differing stages of a cellexpansion phase.

System 200 also includes an optimized cell formulation chamber 210fluidly communicating with first cell suspension chamber 203 via conduit211, optional valves (not shown), and associated pump 212. Such valvesand/or pump 212 may be under the control of process controller 229 asdiscussed in greater detail hereinbelow. Thus, after any initialprocessing in chamber 203, the cell suspension composition can betransferred to the chamber 210 for further processing via the operationof pump 212 to transfer the suspension through conduit 211. A device 213for determining the concentration of cells in the optimized cellformulation within chamber 210 is provided. Device 213 can be of similardesign to device 204 discussed above. A conduit 214 can serve in thetransfer of a sample of the cell suspension composition from chamber 210to the device 213 for analysis. A conduit 215 can optionally be providedfor returning the sample to the chamber 210 should that be appropriate.As above, alternatively, the sample can be discarded after the analysisvia a waste line.

A formulation media unit 216 is fluidly coupled to chamber 210 viaconduit 217. Transfer of materials from one or more chambers of unit 216to the chamber 210 can be facilitated by optional valves (not shown) andpump 218. Such valves and/or pump 218 may be under the control ofprocess controller 229 as discussed in greater detail hereinbelow. Theformulation media unit 216 can contain materials that are to be combinedwith the cell suspension and that are physiologically acceptable foradministration to patients. The formulation media may for exampleinclude saline, cell culture media, drugs such as antibiotics,substances designed to illicit a specific cellular response such asdifferentiation or quiescence, or other materials. In one use, device213 is used to determine the cell concentration of the cell suspensionwithin chamber 210. If the concentration of cells is higher than thatdesired for application to a cell growth substrate, formulation mediacan be added from chamber 216 to dilute the cell suspension to achievethe desired cell concentration. The volume of formulation media to addcan be calculated based upon the overall volume of the compositionwithin chamber 210 and the cell concentration determined by device 213.Beneficial cell concentration values for the optimized cell formulationin chamber 210, to be applied to the cell growth substrate, can rangefrom about 10⁵ to about 10⁸ cells per ml, although other concentrationscan be used. In alternative situations in which the cell concentrationis lower than desired, then chamber 210 can be operably associated withmeans for increasing the cell concentration, e.g. by removing amounts ofmedia. In one embodiment, an outlet 238 from chamber 210 can be providedwith a filter 239 having a pore size selected to prevent passage of thecells but allow passage of the fluid media suspending the cells.Optional valves (not shown) and a pump 240 can be provided to drive orpull amounts of the cell suspension against this filter to selectivelyremove amounts of the fluid media from chamber 210 and thus concentratethe cell suspension within chamber 210. A vibration device 241 can beprovided if needed, to impart vibration to the filter to resist cloggingof the filter with cells during operation. Additionally oralternatively, the system can be configured with appropriate pump(s)and/or valves to direct a reverse flow of liquid through the filter toclean the same in a back flush mode. Such operations and any attendantvalves, pump 240 and/or vibration device 241 may be under the control ofprocess controller 229 as discussed in greater detail hereinbelow.Materials removed from chamber 210 via outlet 238 can for example go toa waste unit “W”. If desired, waste unit W can also be fluidly coupledto chamber 203 or other fluid sources in system 200, to receive wastetherefrom into a single waste chamber or separate waste chambers. Afterupward or downward adjustment of the cell concentration within chamber210 as a result of a prior-initiated concentration measurement orotherwise as a result of the introduction or withdrawal of media fromchamber 210, an additional sample can be drawn off into device 213 andassessed to confirm that the cell suspension is within a desired cellconcentration range. Additional adjustments can be made as necessary toachieve the desired cell concentration. After the cell suspension isconfirmed to have the desired cell concentration, the cell suspensioncan be transferred from chamber 210 to an application device 221 viaconduit 219, using optional valves (not shown) and powered by pump 220.Such valves and/or pump 220 may be under the control of processcontroller 229 as discussed in greater detail hereinbelow.

Application device 221 can be any of a variety of devices for applyingthe cell suspension composition to a cell growth substrate 223 receivedwithin seeding chamber 222. Application device 221 may be stationaryduring operation, or may be movable during operation to apply materialacross a broader area or volume that would be possible if it werestationary. Application device 221 may be under the control of processcontroller 229 as discussed in greater detail hereinbelow. Applicationdevice 221 may in one embodiment be a spray device, for example a spraynozzle or a jet spray apparatus similar to that found in ink jetprinters. Such spray devices can be arranged to apply the cellsuspension composition to the substrate by spraying the compositionagainst the substrate, either from a stationary position or uponmovement in one, two or three dimensions. In another embodiment, theapplication device 221 can be a cannulated device having at least onecannula and preferably a plurality of cannulae for application of thecellular composition to the surface of and/or within the cell growthsubstrate 223 from a stationary position or during movement in one, twoor three dimensions. Some embodiments incorporate a cannulatedapplication device 221 that has one cannula or a plurality of cannulaeinserted into the substrate 223 during application of the cellularcomposition, for example by expressing the cellular composition from thecannula(e) during a pull back operation beginning with the cannula(e)inserted at a distal position within the volume of the substrate 223. Ina specific embodiment, such an application device includes a linear orother array of needles for application of the cell composition to thesubstrate 223. Still other application modes will be apparent to thoseof ordinary skill in the art.

Application device 221 desirably applies the cellular composition to thesubstrate substantially evenly to the surface of the substrate orthrough the volume of the substrate. However, in other embodiments,application device 221 may selectively seed certain portions of thesubstrate, and thus the application device 221 can be configured toapply the cellular composition to specific regions of the substrate 223while leaving other regions of the substrate free of the cellularcomposition. Combinations of different types of application devices 221can be incorporated into system 200, for example wherein the systemincludes both a spray application device and a cannulated applicationdevice.

In particular embodiments of the invention, system 200 has anapplication device 221 configured to seed cells onto and/or into cellgrowth substrates that define internal passages for receiving flow ofcellular compositions, for example substrates such as those depicted inFIGS. 1-8. Application device 221 in such embodiments desirably includesat least one cannula and preferably a plurality of cannulae. In certainembodiments, the cannula(e) are fluidly coupled to the defined passagesin the substrate (e.g. passages 22, 68, 83, 84, 96, and/or 116 of FIGS.1-8) and operable to pass amounts of the cellular composition into thepassages. In additional embodiments, a cannula is provided on theapplication device 221 for each opening of a passage to the exterior ofthe cell growth substrate, wherein the cannulae correspond to andregister with such openings. In application devices 221 that are staticduring application of the cellular composition, the cannula(e) candirect the cellular composition into the passages under pressure, whichwill lead to flow of the cellular composition through the passages, outof the sidewalls of the passages, and into the surrounding volume ofmaterial of the substrate. With cell growth substrates that includeindividual passages that have openings at spaced locations on thesubstrate, for example passages 22 of the substrate of FIG. 4 withspaced openings 56 and 57, passages 22 and 68 of the substrate of FIG. 5with spaced opening pairs 66,67 and 69,70, respectively, passages 83, 84and 22 of the substrate of FIG. 6 with spaced openings 84 and 85, andpassages 96 and 116 of the substrates of FIGS. 7 and 8 with spacedopening pairs 97,98 and 117,118, respectively, application device 221can have a cannula fluidly connected to each opening. Such anapplication device 221 can for example be operated in a flow-throughmode, an opposed flow mode, or a combination thereof. In a flow-throughmode, input cannula(e) force the cellular composition into a firstopening to a passage and output cannula(e) receive fluid that traversesthe passage, having deposited at least a portion of the cells of theinput composition in the cell growth substrate. In an opposed flow mode,input cannula(e) force the cellular composition into both the firstopening and the second opening to a passage, wherein the respectiveinput flows to the passage oppose one another. This can create pressurewithin the passage and thus drive cellular fluid out of the passage andinto the surrounding volumes of substrate material, facilitating a moreeven seeding operation.

A flow-through mode as noted above can involve only a single passage ofthe cellular composition through the substrate, but in advantageousembodiments will be conducted so as to pass the material received in theoutput cannula(e) back through the substrate passage(s) at least onetime to deposit additional cells, which can be accomplished for exampleby collecting the output fluids and reversing the direction of flowthrough the passages, and/or by providing a recirculation loop in whichthe output fluids are directed again to the same input cannula(e) andback through the substrate passage(s) to the output cannula(e). In manyembodiments, the original output fluids, containing unseeded cells, willbe passed back through the substrate passages a plurality of times toseed a higher percentage of the original cells in the cellularcomposition into the substrate.

In this regard, with reference to FIG. 9A, in conjunction with FIG. 9,shown is one embodiment of a circulation loop application device 221useful for seeding an internally-plumbed cell growth substrate 50 suchas that shown in FIG. 4. Application device 221 has a plurality of inputcannulae 221 a and a plurality of output cannulae 221 b, with the inputcannulae 221 a fluidly coupled to openings 56 of substrate 50 and theoutput cannulae 221 b fluidly coupled to openings 57 of substrate 50.Input cannulae 221 a are mounted in input manifold 221 c, which definesan internal input manifold passage 221 d having an opening 221 e, withthe manifold passage 221 d fluidly coupled to and feeding each of inputcannulae 221 a. Opening 221 e is fluidly connected to conduit 219 (FIG.9) which provides a feed of the cellular composition from chamber 210.Output cannulae 221 b are mounted in output manifold 221 f, whichdefines an internal output manifold passage 221 g having an outputopening 221 h, with the output cannulae 221 b fluidly coupled to andfeeding output manifold passage 221 g. A circulation loop 221 i isprovided fluidly connecting input opening 221 e of input manifold 221 cand output opening 221 h of output manifold 221 f. A cell counter 221 j,such as one using the Coulter principle, light scattering or flowcytometry to provide a calculated cell concentration, can be fluidlycoupled to circulation loop 221 i. Cell counter 221 j can determine cellconcentration in the fluid circulating in loop 221 in-line or by drawingand assessing a sample, as noted in the discussions above. Optionalvalves (not shown) and a pump 221 k is provided to drive circulation inthe application device 221 for seeding the substrate 50. Such cellcounter 221 j, valves, and/or pump 221 k may be under the control ofprocess controller 229 as discussed in greater detail hereinbelow.

In use, a cellular composition fed to the circulation loop ofapplication device 221 from conduit 219 in a batch operation,continuously, or intermittently, is repeatedly circulated through inputmanifold 221 c, input cannulae 221 a substrate 50, output cannulae 221b, output manifold 221 f and circulation loop 221 i. During thisoperation, cells will be seeded into substrate 50, and some cells willremain in the circulating fluid. Cell counter 221 j can be used tocontinuously or periodically determine the cell concentration of thefluid circulating in circulation loop 221 i. Seeding of an acceptablepercentage of the originally-provided cells into substrate 50 will bedetermined by system 200 and/or signaled to a user, for example by useof process controller 229 described in greater detail hereinbelow, at apoint in time when the cell concentration detected by cell counter 221 jreaches a desired or predetermined low value. Circulation withinapplication device 221 can thereafter be terminated, and substrate 50can be prepared for administration to the patient immediately, or aftera further incubation or culture period.

As disclosed above, in other embodiments, application device 221 isconfigured to move during application of the cellular composition to thesubstrate. With reference to FIG. 9B, in conjunction with FIG. 9, shownis one such application device 221, useful for applying a cellularcomposition to a pre-plumbed cell growth substrate such as substrate 20of FIG. 3 or substrate 50 of FIG. 4 (shown in phantom, dotted lines ofFIG. 9B). Application device 221 of FIG. 9B includes a plurality ofcannulae 221 k mounted to an input manifold carriage 221 l. Inputmanifold carriage 221 l defines an internal manifold passage 221 mhaving an input opening 221 n, with manifold passage 221 fluidly coupledto and feeding cannulae 221 k. Input opening 221 n is fluidly connectedto conduit 219 of system 200, which feeds the cellular composition fromchamber 210. Input manifold carriage 221 l is attached to carriage posts221 o, which are translatably received through openings in post feedmounts 221 p. An electric motor, solenoid, or other powered mechanism(not shown) is mechanically coupled to posts 221 o and operable towithdraw posts 221 through mounts 221 p, preferably at a constant rateor in another predetermined manner, thereby causing a pull-back motionof the manifold carriage 221 l and its associated cannulae 221 k. Thesubstrate 20,50 is held stably in its starting position during thispull-back operation by clamping members 224 a engaging at least portionsof the periphery of the substrate 20,50, or any other suitable mechanismfor maintaining a registration position of the substrate relative toapplication device 221 (see additional discussions below pertaining toregistration devices 224). With cannulae 221 k initially received withinpassages 22 of substrate 20 or 50 with their distal tips locateddistally therein, an automated pull-back motion can be initiated duringwhich the cellular composition is pumped and expressed from the tips ofcannulae 221 k at a predetermined rate. In this fashion, by controllingthe rate of pull-back, and the rate of flow from cannulae 221 k, system200 can highly effectively seed the cell growth substrate with a givenflowable cellular composition.

With continued reference now to FIG. 9, as discussed above, system 200can include a registration device 224 for retaining the cell growthsubstrate 223 in a predetermined position relative to a starting orin-process position of application device 221, or otherwise withinchamber 222. In this fashion, device 221 can be reliably operated todeposit the cellular composition to the substrate 223 in a desiredmanner. Registration device 224 can for example include upstandingwalls, clamps, pins, or other features that associate with the materialor shape of substrate 223 to hold the same in position within thechamber 222. Further, substrate 223 can be equipped with mechanicalelements that cooperate with registration device 224 during loading intochamber 222. For example, substrate 223 could have temporarily(non-implantable) or permanently attached (implantable) strips or barsof relatively rigid plastic for reliably cooperating with a rigidstructure of registration device 224 to stably position substrate 223within chamber 222. After seeding and before implantation, suchmechanical elements can be removed from substrate 223 if they are notintended or suitable for implant.

System 200 can also have a distribution assist device 225 forfacilitating the distribution of the cells across or through thesubstrate 223. Distribution assist device 225 can therefore be operableto impart a force to the cellular composition within chamber 222 and/orresident upon substrate 223. The distribution assist device 225 may forexample be operable to create a vacuum to pull the cellular compositionand thus cells through the thickness of the substrate 223, to moreevenly distribute the cells within substrate 223. Alternatively,distribution assist device 225 can be a magnet, such as a permanent orelectromagnet, and can impart a magnetic field encompassing substrate223. This, in conjunction with magnetic materials associated with thecells, for example, magnetite liposomes to which the cells are attached,can be used to drive the cells through and/or along the substrate 223 todistribute the cells. Alternatively, distribution assist device 225 canbe operable to impart motion to the substrate 223 such as vibration orrotation, to assist in distributing the cell composition through thesubstrate 223. In one embodiment, distribution assist device 225 canimpart rapid rotation so as to drive the cellular composition bycentripetal force, and the substrate 223 can be arranged in the path ofthe driven cellular composition (e.g. along a substantially vertical orotherwise upstanding wall when the rotation is in the horizontal plane).Using this arrangement the cellular composition can be driven againstand into the substrate 223. Still other devices 225 that impart forcesto the cellular composition and/or to the substrate 223 to enhance thecombination of the two can be used, including for example devices 225that can impart pulsatile and/or bidirectional flow to liquids withinchamber 222. In certain embodiments, a sheet form substrate 223 can bemounted within chamber 222 so as to divide the chamber 222 into firstand second volumes, the first and second volumes fluidly sealed from oneanother except across the substrate. Directional flow can then be causedwithin chamber 222 by creating a pressure differential with the aid ofdistribution device 225 so as to provide transmural flow of the cellsuspension across the substrate 223 to distribute cells onto andpotentially also into the substrate 223. All functions of chamber 222may be under the control of process controller 229 as discussed ingreater detail hereinbelow.

In other embodiments, an extracellular matrix hydrolysate or gelmaterial, including for example those described herein, can be chargedinto chamber 222 along with the cells, and a solid, three-dimensionallystable cell growth substrate can be incubated in a volume of thehydrolysate/gel and cell mixture, including while completely immersedtherein. Agitation of the combined materials can be conducted asdescribed herein. The solid, three-dimensionally stable substrate can bean ECM material as described herein, preferably retaining at least onenative (endogenous) bioactive component or a combination thereof asdescribed herein, and/or can be in the form of a sheet, filament orthread, or particulate. The combined materials can be incubated asdescribed herein and thereafter administered to the patient.

In still further embodiments, an application device 221 can have one ormore cannula(e) as described herein, and the outlet opening(s) of thecannula(e) can be positioned internally within the volume of a solidcell growth substrate 223, such as a sponge, foam or other similarfibrous substrate. The cellular fluid can then be injected into theinner volume of the substrate material 223 and can migrate underpressure toward (and potentially out of) the exterior surfaces of thesubstrate 223, depositing cells within the substrate on the way. Incertain embodiments, the cannula(e) and its/their openings and substrate223 can be configured to cause a substantially even flow of the fluidmaterial through the substrate 223 in all directions from a generallycentral location within the substrate, so as to facilitate an evendistribution of cells within the substrate 223.

During and/or after seeding of a substrate 223 and incubation for a timeas described herein, system 200 can assess the seeded substrate 223 todetermine whether substantial numbers of non-adhered cells remain on orin substrate 223. This can be achieved, for example, by flushing all ora portion of substrate 223 with a probing pulse of liquid to dislodgenon-adhered or poorly-adhered cells, and the liquid pulse then collectedand assessed for the presence of cells, including in some embodimentsquantitatively. The probing pulse can for example be fed to chamber 222via line 227 from a chamber in media unit 226 (discussed further below),or another independent source and feed line could be provided. The testpulse liquid can for example be physiological saline, preconditioningmedia, cell culture media, or any other liquid allowing for thedisplacement and subsequent detection of non-adhered or poorly-adheredcells on the substrate 223. In one embodiment, the collected pulsevolume with the dislodged cells can be directed to detector 213 asdiscussed herein, e.g. a cell counting device utilizing the Coulterprinciple or another means for counting cells. If an overly high numberof cells are dislodged by the probing pulse, the substrate 223 and cellscan be incubated for a further period of time to allow for cellattachment. If no cells or a sufficiently low number of cells arecollected in the test pulse, then the seeded substrate 223 can beremoved from chamber 222 and administered to the patient. System 200 canoptionally generate a signal, such as a visual and/or audible signal, toindicate that the seeded substrate 223 within chamber 222 is ready foradministration to the patient. All of these operations can be under thedirection of a process controller 229 discussed further below.

System 200 can also include a preconditioning media unit 226 having oneor multiple chambers fluidly coupled to application device 221 viaconduit 227, and powered by pump 228. Application device 221 can shareduties in applying the cellular composition and preconditioning media tothe substrate 223, or separate application devices can be incorporated.Preconditioning unit 226 can contain and supply media for pretreatingthe substrate 223 to condition the same for receipt of the cellularcomposition. The preconditioning media within chamber 226 can, forexample, be a cell culture medium, or can contain proteins or othersubstances beneficial to the cells or which render the substrate 223more compatible with survival of the cells. In one embodiment, thepreconditioning media in unit 226 includes serum, preferably autologousserum from the patient to receive the cellular graft produced usingsystem 200. The preconditioning media can be applied to the substrate223 using the application device 221. Optionally also, preconditioningunit 226 or other components of system 200 can incorporate materials forrinsing the substrate 223 after treatment with other preconditioningmedia, for testing the substrate, or other operations. For thesepurposes chamber 222 can include a drain connected to a waste line incertain embodiments.

System 200 is preferably automated and thus includes a processcontroller 229 which is operable to control the various mechanisms insystem 200 such as pumps, valves, temperature control units, detectorssuch as devices 204 and 213, the application device 221, thedistribution assist device 225 and other components of system 200, toachieve the functions herein recited. Process controller 229 may be aprocessor-based system for controlling, either completely automaticallyor by assisting user control, all of the various mechanisms of system200. Although not illustrated in FIG. 9 for the sake of clarity, processcontroller 229 is coupled to the various mechanisms of system 200 thatit controls or from which it receives sensed data by means ofappropriate input and/or output communication paths, as will be evidentto those skilled in the art.

Process controller 229 may include electronic user input device(s) 230,such as a keyboard and/or a mouse, connected via connection 231. Adisplay 232, connected via connection 233, may be provided to displaystatus entries, results of analyses, prompts to the user, and otherinformation as desired. A printer device 242, connected via connection243, may be provided to produce a printed record of results of analyses,current status of the cell seeding operation, and/or a record of theoperations completed by the system 200 during a cell seeding operation,and other information as desired. These devices are coupled to allow theinput of user control instructions into the process controller 229 sothat the system 200 may be operated as needed for the current cellseeding operation, and various forms of information may be displayed,printed or manipulated by users.

The process controller 229 may be implemented on a personal computer, aworkstation computer, a laptop computer, a palmtop computer, a tabletcomputer, a wireless terminal having computing capabilities (such as acell phone or personal digital assistant (PDA) having a Windows CE, aPalm operating system, or the like), or with a microcontrollerintegrated into the system 200. It will be apparent to those of ordinaryskill in the art that other computer system architectures may also beemployed, and that the particular architecture chosen is not critical tothe presently disclosed systems and methods.

In general, such a process controller 229, when implemented using acomputer, comprises a bus for communicating information, a processorcoupled with the bus for processing information, a main memory coupledto the bus for storing information and instructions for the processor, aread-only memory coupled to the bus for storing static information andinstructions for the processor. The display 232 is coupled to the busfor displaying information for a user of system 200 and the inputdevice(s) 230 is coupled to the bus for communicating information anduser command selections to the processor. A mass storage interface forcommunicating with a data storage device containing digital informationmay also be included in process controller 229, as well as a networkinterface for communicating with a network.

The processor may be any of a wide variety of general purpose processorsor microprocessors such as the PENTIUM, CORE and XEON microprocessorsmanufactured by Intel Corporation, a POWER PC or POWER ISA manufacturedby IBM Corporation, a SPARC processor manufactured by Sun Corporation,or the like. It will be apparent to those of ordinary skill in the art,however, that other varieties of processors may also be used in anyparticular computer system. Display 232 may be a liquid crystal device(LCD), a cathode ray tube (CRT), a plasma monitor, a light emittingdiode (LED) device, or other suitable display device. The mass storageinterface may allow the processor access to the digital information onthe data storage devices via the bus. The mass storage interface may bea universal serial bus (USB) interface, an integrated drive electronics(IDE) interface, a serial advanced technology attachment (SATA)interface or the like, coupled to the bus for transferring informationand instructions. The data storage device may be a conventional harddisk drive, a floppy disk drive, a flash device (such as a jump drive orSD card), an optical drive such as a compact disc (CD) drive, digitalversatile disc (DVD) drive, HD DVD drive, BLUE-RAY DVD drive, or anothermagnetic, solid state, or optical data storage device, along with theassociated medium (a floppy disk, a CD-ROM, a DVD, etc.)

In general, the processor retrieves processing instructions and datafrom the data storage device using the mass storage interface anddownloads this information into random access memory for execution. Theprocessor then executes an instruction stream from random access memoryor read-only memory. Command selections and information that is input atinput device(s) 230 are used to direct the flow of instructions executedby the processor. The results of this processing execution may then beused to control the various mechanisms in system 200.

The process controller 229 is configured to generate an output fordisplay on the display 232 and/or for driving the printer 242 to print ahardcopy. Preferably, the display 232 is also a graphical userinterface, allowing the user to interact with the displayed information.

The process controller 229 may also be configured to communicate withone or more external systems (not shown) via a network, such as a localarea network (LAN), a wide area network (WAN) or the internet. Both theprocess controller 229 and the external systems may be configured to actas a web server, a client or both and may be browser enabled. Thus, thesystem 200 may access and/or store information remotely, may becontrolled and/or monitored remotely, and data exchange between processcontroller 229 and other systems may occur.

To minimize loss of cells during transfer operations, it may bedesirable to utilize as few chambers as possible in the processing ofthe cell suspension. Thus, in certain embodiments, system 200 can omitoptimized cell formulation chamber 210 and its associated detector 213,and instead process the cell suspension to a desired condition forapplication to the substrate within first chamber 203. Thus, shown inphantom is conduit 234 in this alternate embodiment, fluidly connectingthe chamber 203 to the application device 221. Conduit 234 can transferan optimized cell suspension to device 221 under the power of pump 235.In this more simplified version, system 200 can also include conduit 236feeding from formulation via chamber 216 to cell suspension chamber 203,and powered by pump 218. Accordingly, the cell suspension compositioncan be processed with processing media from chamber 207, and withformulation media from chamber 216, all within a single chamber 203. Forsubsequent processing steps with various media, chamber 203 can beequipped with means for removing applied media, such as filters asdiscussed above, or other devices that can remove liquid or otherwiseconcentrate the cells after the application of volumes of treatmentmedia from chambers 207 and/or 216. After processing and adjustment ofthe cell suspension composition within chamber 203, device 204 can beused to confirm that the cell suspension composition has a cellconcentration within a desired range, whereafter the composition can beadvanced to application device 221 for application to the substrate 223.For convenience in handling and operation, system 200 can include ahousing 237 housing some or all of the components discussed herein.

Referring to FIG. 10, shown is a schematic diagram of another automatedcell seeding system 250. System 250 can be used to prepare a flowablecellular graft, which in certain embodiments can be delivered byminimally-invasive techniques, such as through needles, catheters, orother percutaneously-introduced delivery devices. System 250 includesmany components in common with system 200 (FIG. 9) described above,which are identically numbered in FIG. 10 and need not be describedagain here. As to other components, system 250 includes a cell growthsubstrate input chamber 251 for receiving a particulate-form substrate,a gel-form substrate or precursor materials thereto, or a combination ofthese, potentially also along with other materials for the graft. Theprocessed cell suspension composition is combined with the cell growthsubstrate in chamber 251 via conduit 219 using optional valves (notshown) and under the power of pump 220. A prepared composition fromchamber 251, including the cells and the substrate material(s), istransferred by flow via conduit 252 to a receptacle 254, using optionalvalves (not shown) and powered by pump 253. Such valves and/or pumps 220and 253 may be under the control of process controller 229. Receptacle254 can be a chamber device temporarily attached and fluidly connectedto conduit 252, which can be removed to transport the cellular graftmaterial away from system 250. Receptacle 254 can thus be a vial, bag,or other container fluidly connected to conduit 252. In certainembodiments, receptacle 254 can comprise a delivery device fordelivering the cellular graft composition to the patient, or a componentthereof. In one embodiment, receptacle 254 includes a chamber,illustratively a syringe barrel, from which the cellular graftcomposition will be forcefully expelled for delivery to patient tissueby a fluidly coupled needle, catheter, or other minimally-invasivedevice. System 250 can include structural mating features for insertionand mating of such a receptacle 254 in a fashion that fluidly couples toconduit 252 for receipt of a prepared, flowable cellular graft material.

Chamber 251 can be equipped with a device 255 that mixes materialswithin chamber 251. Device 255 can be comprised of a mechanism locatedcompletely external of chamber 251, but which imparts motion to chamber251 or parts thereof to mix components therein. For example, such adevice can comprise a vibrator, a shaker, a rotary element such as avortexer, or a static mixer positioned in a flow path for the componentsto be mixed. Device 255 can also be comprised of a mechanism locatedwithin chamber 251 such as a paddle, a stir bar, or impeller, suchmechanism driven by a motor or other means, so as to agitate flowablecompositions received within chamber 251. Device 255 can also becomprised of components both internal and external of chamber 251, suchas an external moving (e.g. rotating) magnet that drives amagnetically-coupled paddle, stir bar, or other element within chamber251. Device 255 may be under the control of process controller 229.

In addition to providing mixing within chamber 251, system 250 caninclude a thermal control device 256 associated with chamber 251 and/ora thermal control device 257 associated with receptacle 254, operable tocontrol the temperature of respective materials therein. Thermal controldevices 256, 257 may be under the control of process controller 229, andmay contain appropriate temperature sensing elements to provide currenttemperature feedback readings to facilitate the regulation oftemperature. Thermal control device 256 can be a heating element and/orcooling element. In certain modes, thermal control device 256 can beoperated to control the temperature of a material within chamber 251 andthereby regulate its viscosity. Illustratively, the cell growthsubstrate composition received within chamber 251 may comprise agellable material, and thermal control device 256 can be operated tomaintain the material in an ungelled state prior to and during itscombination with the cell suspension fed from conduit 219. In thismanner, the less viscous state of the substrate material will allow amore facile mixing with the cell suspension material. In this use, forgellable materials that increase in viscosity with decreasingtemperature, thermal control device 256 will be operated so as to heatthe materials within chamber 251. In this regard, the heating willdesirably be conducted to avoid any significant thermal damage to thecells, for example heating to a temperature of less than about 42° C.when cells are present. In certain embodiments, the gellable materialwithin the substrate composition will be selected so that it gels at atemperature of 37° C. or slightly above. For mixing with the cellsuspension to achieve a substantially homogenous composition, thematerial can be heated at a temperature above its gel point, but below atemperature at which the cells would be significantly damaged (e.g.below about 42° C.). After mixing, the cell/substrate composition can bemaintained above the gel temperature of the substrate material prior toand during delivery to a patient, for example using thermal controldevice 257. In this fashion, a less viscous (and more flowable) deliverystate is provided during delivery, e.g. through a needle or othercannula, but the material will gel and thus increase in viscosity afterdelivery to the patient (e.g. a human patient having a normal bodytemperature of about 37° C.) to facilitate maintenance of the deliveredgraft material at the site of administration. Alternatively, aftermixing to provide substantial homogeneity, the cell/substrate materialcan be allowed or caused to gel external of the patient (e.g. by coolingwith device 256 and/or 257), and can then be delivered as a more viscousgraft.

For gellable substrate materials that gel or otherwise increase inviscosity with increasing temperature, thermal control device 256 can beoperated so as to cool the materials within chamber 251. In this regard,the cooling will desirably be conducted to avoid any significant freezedamage to the cells, for example cooling to a temperature of greaterthan about 0° C. In certain embodiments, the gellable material withinthe substrate composition will be selected so that it is gelled orotherwise increases in viscosity at a temperature of about 37° C., ascompared to lower temperatures, for example about room temperature(about 25° C.) or below. For mixing with the cell suspension to achievea substantially homogenous composition, the material can be cooled withdevice 256 to retain a less viscous condition. After mixing, thecell/substrate composition can be maintained in a cooled state usingthermal control device 256 and/or 257 prior to and during delivery. Inthis fashion, a more flowable state is provided during delivery, e.g.through a needle or other cannula, but the material will increase inviscosity after delivery to the patient (e.g. a human patient having anormal body temperature of about 37° C.) to facilitate maintenance ofthe delivered graft material at the site of administration.Alternatively, after mixing to provide substantial homogeneity, thecell/substrate material can be warmed, e.g. using device 256 and/or 257,or allowed to warm in an ambient setting, for delivery as a more viscousgraft.

Conditions other than temperature can also be used to cause cell growthsubstrate materials to gel when desired (e.g. after a mixing operationwith cells). For example, collagenous materials can be selected that gelwith increasing pH. During a mixing operation within chamber 251, the pHof the substrate/cell composition can be maintained at a relativelylower level at which the composition remains more flowable, but at whichthe cells can survive, at least during the period of mixing.Illustratively, such a pH may be in the range of about 4 to about 6.5.After mixing, the pH of the composition can be increased so as to causethe collagenous material to gel and increase the viscosity of the graftmaterial. Such a pH adjustment may be accomplished by adding abiologically compatible base or buffer, or both. These additives may beprovided from a chamber in unit 226, or another reagent chamber inprovided in system 250. The post-mixing pH of the composition can, forexample, be in the range of above about 6.5 up to about 8. Desirably,the post-mixing pH will be one which is suitable for administration to apatient, which in certain specific embodiments will be about 7 to about7.5.

After combination of a cell growth substrate material with a cellularcomposition in system 200 or 250 discussed above, the seeded graftmaterial can be immediately administered to a patient. In otherembodiments, the cell seeded graft material can be incubated ex vivo forat least a period of time sufficient to achieve attachment of at leastsome of the cells to the cell growth substrate, and desirably asubstantial percentage of cells, for example greater than about 20% ofthe originally-provided cells. Such cell-attachment incubation phasescan have a duration of one minute up to about five hours, and inparticular embodiments about five minutes up to about three hours.During this period, the composition will be maintained under conditionsin which cells in the composition, at least in part through their nativecapacity to attach to surfaces, attach to cell growth substrate sheets,particles or other material(s) in the combined composition. Cellattachment phases or cycles can be conducted during which no significantexpansion of the number of originally-provided cells is experienced, forexample wherein the composition has no more than ten percent greaterviable cells than those originally combined with the cell growthsubstrate, and in certain embodiments wherein the composition asessentially the same number of cells or fewer cells than the numberoriginally combined with the cell growth substrate.

In the preparation of flowable graft materials in automated system 250,the composition can optionally be continuously or periodically agitatedduring a cell attachment phase as discussed above, for example usingmixer device 255. In particular embodiments, mixer device 255 will beused to periodically gently agitate the composition during a cellattachment cycle, with relatively static cell attachment phasesoccurring between agitation phases. For example, multiple non-agitatedphases may have a duration of about three minutes to about twentyminutes each, interrupted by shorter agitation phases, for example ofabout ten seconds to five minutes, performed gently so as to bringadditional suspended free cells into contact with cell growth substrateparticles or material but avoid significant dislodgement of cellsalready attached to substrate materials.

During and/or after seeding of a substrate and incubation for a timewithin chamber 251 as described herein, system 250 can assess a flowablesubstrate cell/substrate mixture to determine whether substantialnumbers of non-substrate-adhered cells remain in the suspending mediumof the mixture. This can be achieved for example by filtering the sampleupon collection to remove the substrate material with adhered cells,leaving only cells freely suspended in the suspending medium. The freesuspended cells can then be routed to detector 213 or anotherindependent detector, and the cells counted utilizing the Coulterprinciple, light scattering or another means for counting cells asdiscussed herein. In another mode, the entire medium from chamber 251can be sampled and tested (e.g. including particulate substrate,suspending medium and cells) utilizing the Coulter principle, lightscattering or another means. The assessed values for the medium (e.g.scattering or electrical resistance) will vary in accordance with whatpercentage of the cells are adhered (e.g. to substrate particles) versusfreely suspended, and thus the assessed values can be correlated to anacceptable level of cellular adherence to the substrate. If an overlyhigh number of cells remain free in the suspending medium, the substrateand cells can be incubated for a further period of time to allow forcell attachment. If no cells or a sufficiently low number of cellsremain free in the suspending medium, then the seeded substrate materialcan be removed from chamber 251 and administered to the patient. As withsystem 200, system 250 can optionally generate a signal, such as avisual (e.g. on display 232) and/or audible signal, to indicate that theseeded substrate material within chamber 251 is ready for administrationto the patient. All of these operations can be under the direction ofprocess controller 229.

In systems 200 and 250 disclosed above, the recited chambers can beprovided by any suitable arrangement including for example bags, vials,passages, plastic containers, or the like. The recited conduits can beprovided by appropriate tubing, lumens occurring through larger plasticstructures of the system, or any other suitable arrangement. As well, itwill be understood that valves can be provided within the chambersand/or the conduits, to coordinate with pumps or other material transfermeans to selectively permit or prevent flow as appropriate to thecircumstance. These and other physical system features will readilyoccur to those skilled in the art given the disclosures herein.

Systems 200 and 250 can be configured to provide a relatively shortresidence time for the cellular graft materials prior to implantationinto a patient, for example up to about three hours. Such configurationswill typically be designed to achieve attachment of the cells to asubstrate, without any significant expansion of cell numbers (e.g. noexpansion, or less than a 10% increase in cell numbers). However,systems 200 and 250 can in certain embodiments be configured for longerterm incubation and culture of the cellular graft materials to achieveexpansion of the number of cells as compared to the number originallyseeded onto the substrate, for example greater than a 20% increase inthe number of cells, and in some embodiments at greater than a 100%increase. For these purposes, systems 200 and 250 can also includemechanisms for imparting forces, for example shear forces, strain, ortensile forces, to the substrate during the culture period. These forcescan impact the growth and differentiation of the seeded cells during theincubation/culture period, and lead to protein expression patterns thatdiffer relative to the same cells if incubated/cultured in the absenceof the forces. In this manner, the cellular grafts can be enhanced to agiven end use in a patient.

As well, in certain embodiments, system 200 or 250 can include asecondary cell incubation chamber isolated from chamber 222. Cells thatdiffer from those incubated in chamber 222 can be cultured in thesecondary chamber so as to secrete signaling molecules such as hormones,cytokines, growth factors, or others, which are transferred to chamber222. The signaling molecules can contact the cells under culture inchamber 222, for example to modulate their growth or differentiation.Transfer of the signaling molecules from the secondary incubationchamber to chamber 222 may for example be accomplished by pumping themthrough a conduit, by flow across a membrane, or other means. In someforms, the signaling molecules can be effective to drive a higherpercentage of stem or progenitor cells cultured in chamber 222 down agiven differentiation pathway. Control of the secondary cell incubationchamber may be by means of process controller 229 or by means of aseparate process controller optionally in communication with processcontroller 229, as discussed hereinabove.

Particulate Multicellular/Substrate Graft Materials

In certain inventive embodiments, a flowable cellular graft material isprovided that includes multicellular bodies suspended in a liquidmedium, wherein the bodies are each comprised of a cell growth substrateparticle having cells adhered thereto. The substrate particles can havea maximum cross sectional dimension of about 20 microns to about 2000microns, and in certain embodiments about 100 to about 1000 microns. Thesubstrate particles can be substantially uniform in size relative to oneanother, e.g. having maximum cross sectional dimensions within about20%, or 10%, of one another, or can vary in size with respect to oneanother (e.g. having some smaller particles and some larger particles,potentially a controlled overall population created by mixing two ormore substantially uniform particle populations, where the populationsare of different sizes relative to one another). In advantageousvariants, the substrate particles are in sheet form, and can have asheet thickness of about 20 to about 2000, more preferably about 20 toabout 500 microns, and/or a maximum cross sectional axis lengthconsidered in the plane of the sheet (e.g. height or width) that isgreater than the sheet thickness and in the range of about 25 to about2500 microns, more preferably about 100 to about 1000 microns. The sheetthickness can be in the range of about 20 to 1000 microns, and/or themaximum cross sectional axis length considered in the plane of the sheetcan be in the range of about 100 to about 1500 microns, in certainembodiments. In addition or alternatively, the substrate particles canbe relatively rounded or compact, as opposed to long and fibrous, whenconsidered in the plane of the sheet. The substrate particles can haveshapes that are regular with respect to one another or which areirregular with respect to one another. In certain embodiment, theparticles can be sheet form particles having a generally circular, ovoidand/or polygonal (e.g. having three to ten sides, e.g. triangular,square or otherwise rectangular, pentagonal, hexagonal, etc.) shape. Forexample, the substrate particles, or a substantial percentage of them inthe composition (e.g. above about 25%), when considered in the plane ofthe sheet, can have a maximum cross sectional dimension axis which is nomore than about two times the length of the cross sectional dimensionaxis taken on a line perpendicular to and centered on the maximum crosssectional dimension axis; preferably, at least about 50% of thesubstrate particles will have this feature, and more preferably at leastabout 70% of the substrate particles will have this feature. Suchparticulate cell growth substrate materials also constitute anembodiment of the present invention, alone (e.g. as cell-free tissuegraft materials) or used in combination with cellular compositions asdiscussed herein.

Small, sheet-form substrate particles as discussed above can be cut fromlarger sheets of substrate material. In certain embodiments, the largersheet of substrate material will be an extracellular matrix sheetmaterial harvested from a tissue source and decellularized, as discussedherein. Sheet-form particles having the above-described characteristicscan be cut from larger ECM sheets using mechanical implements such aspunches or dies, or by cutting using lasers, or using any other suitablemeans. In desired embodiments, the cutting method used will noteliminate the native bioactive ECM character or native bioactive ECMmolecules, as discussed in more detail herein, when this character orthese molecules are resident in a larger starting ECM sheet beingprocessed. Additionally, the ECM sheet being processed, and theresultant ECM sheet particles can have a retained native epithelialbasement membrane on one or both sides of the sheet material, and/orbiosynthetically deposited basement membrane components on one or bothsides of the sheet. To provide native epithelial basement membrane onboth sides of the sheet, two isolated decellularized ECM layers, eachhaving a single basement membrane side and an opposite side, can bestacked and fused or bonded to one another with the basement membranesides facing outwardly. The resulting bilayer sheet can then beprocessed to form the sheet-form particles as described above. Toprepare particles with deposited non-native basement membranecomponents, a decellularized ECM sheet can be conditioned by growingepithelial, endothelial or other cells on both sides to deposit basementmembrane components. The cells can then be removed while leaving thebasement membrane components, and the sheet then processed to preparethe sheet-form particles as described above.

FIG. 11 provides a digital image of an illustrative, very small ECM“dot” that was laser cut from a larger ECM sheet. In this particularillustration, the ECM sheet utilized was single layer porcine smallintestinal submucosa, as available from Cook Biotech Incorporated, WestLafayette, Ind., USA. As can be seen, the length of the maximum crosssectional axis in the plane of the sheet is about 505 microns, whereasthe length of the cross sectional axis on a line perpendicular to andcentered on the maximum cross sectional axis is about 413 microns.Accordingly, these two length dimensions are within about 25% of oneanother, providing a compact sheet particle structure. ECM particulatecompositions having greater than about 50% of the particles exhibitingthis level of correspondence in length dimensions can be especiallybeneficial in use, particularly where greater than about 50% of theparticles have a maximum cross sectional axis in the plane of the sheetin the range of about 100 to about 1000 microns.

To prepare a cell seeded graft composition, particulate growth substrateas described above can be combined with a cellular preparation, forexample using system 250 described hereinabove. For flowable grafts, theparticulate growth substrate can be suspended in a liquid medium, suchas an aqueous medium. Prior to administration, the graft composition canbe incubated during a cell attachment cycle, such as any of thosediscussed above. The size and relatively planar compact shape of theparticulate cell growth substrate provides advantageous suspension andcell attachment characteristics, which can also be enhanced when aflexible substrate material, such as an extracellular matrix sheetmaterial, is used. For administration to the patient, the flowable cellseeded graft can be loaded in a syringe or other delivery device, andthe graft delivered to a tissue targeted for grafting. Illustratively,with reference to FIG. 11, shown is a medical device 300 including aflowable cellular graft composition 301 loaded in a syringe 302.Cellular graft composition 301 includes a plurality of cellularizedbodies 303 that include a matrix particle 304, as discussed here andabove, and a population of cells 305 attached to each matrix particle304. In certain embodiments the cells 305 form a generally confluentlayer of cells covering the matrix particle 304. The cellularized bodies303 are suspended in a liquid medium 306, such as an aqueous mediumoptionally containing nutrients for the cells, and which isphysiologically compatible with a human or other patient. Cellular graftcomposition 301 is flowable and received within the barrel 307 of thesyringe 302. A plunger 308 is received within barrel 307 and operableupon linear actuation to drive composition 301 through the fluidlycoupled needle 309 and out the opening 310 thereof. Medical device 300can therefore be used to administer the composition 301 into tissues ofthe patient. In certain preferred embodiments, the target tissues are inneed of revascularization and the cellular graft bodies 303 includecells 305 capable of forming blood vessels, for example endothelialcells or endothelial progenitor cells, including in certain embodimentsendothelial colony forming cells as discussed herein. Upon injectioninto the target tissue, the matrix particles 304 will assist inretention of the cells 305 in the targeted region. In particularlypreferred embodiments, particles 304 are extracellular matrix particlesas described herein.

Cellular Grafts with Gel-Coated Matrix Substrates

Cellular grafts of the invention can have a porous matrix cell growthsubstrate that is at least in part coated with a gel, such as anextracellular matrix gel, with the cells incorporated within the gel, onthe surface of the gel, or both. The gel can be applied to the porousmatrix substrate before, after, or in admixture with the cells, or anycombination of these. The gel can incorporate substances that enhancethe ability of cells to attach to the substrate, such as fibronectin,laminin, collagen I, or other material(s). When applied in admixturewith cells, the gel, or precursor material(s) to the gel, can be appliedin a less viscous (e.g. ungelled state) to facilitate application to thesubstrate and if desired penetration into the porous network of thesubstrate along with the cells. The gel or gellable precursor(s) canthen be allowed or caused to gel or otherwise increase in viscosity topromote rapid adherence of the gel and cells entrained therein to theporous matrix substrate. In certain embodiments, the gel is comprised ofan extracellular matrix gel, for example as described in United StatesPatent Application Publication No. US20070082060 published Apr. 12,2007, publishing U.S. patent application Ser. No. 10/569,218 filed Aug.25, 2004, which is hereby incorporated herein by reference in itsentirety. Accordingly the gel can include an ECM hydrolysate compositionprepared by digesting ECM tissue such as that described herein with acidand/or enzyme, which composition is gellable upon increasing the pH toabout 6.8 to about 8, and/or upon increasing the temperature of thematerial to about 37° C. Such compositions can contain native collagenand native (endogenous) bioactive non-collagen components of thestarting ECM material such as growth factors, glycosaminoglycans,proteoglycans and/or other materials as discussed in conjunction withECM materials below.

System 200, discussed above, can be used to prepare these cellulargrafts with gel-coated matrix substrates. For example, a gel or gelprecursor(s) can be applied to the substrate 223 using applicationdevice 221. For application of the gel material prior to the cells, thegel or gel precursor can be fed from precondition media unit 226 andapplied to the substrate 223. For combination of the cells with the gelor gel precursor(s) prior to application to the substrate, the cellularand gel materials can be mixed in-line by combining the feeds fromconduits 219 and 227 prior to release from application device 221, orthe cellular material can be combined with the gel or gel precursor(s)within a chamber of precondition media unit 226, or another chamber,prior to feed to the application device 221. Temperature and/or pHcontrol can also be provided by system 220, if needed, to facilitate anincrease in viscosity of the applied material after it is in contactwith substrate 223. Illustratively, for pH adjustment, an amount of abasic substance, such as NaOH, can be combined with the cell/gelprecursor composition immediately prior to application to the substrate223, whereupon the complete gelling of the material will be sufficientlydelayed for application to the substrate 223 and subsequent firming.Alternatively, the substrate 223 can be preconditioned with a basic orbuffer substance to neutralize and increase the pH of a more acidiccell/gel precursor material upon contact with the substrate 223. Stillfurther, the temperature of the cell/gel precursor material can beincreased by a heating element before and/or after application to thesubstrate 223. These and other gel-forming adjustments to conditions canbe automated by system 200.

Cellular Graft Filaments

In additional embodiments, the invention provides cellular grafts whichinclude a cell growth substrate in the form of an elongate filament,with a population of cells attached along the filament. In theseembodiments, the filament can have a length of at least about 1 mm, forexample in certain embodiments in the range of about 1 to about 30 mm,or about 5 to about 30 mm. The filament can also have a greatest crosssectional dimension of about 20 microns to about 2 mm, or about 100microns to about 1 mm. Extracellular matrix substrates as describedherein are preferred for these purposes. To prepare such cellulargrafts, the elongate substrate can be incubated in the presence of acell suspension containing the desired cells for at least a period oftime sufficient for cell attachment to the substrate, for example in anautomated system such as system 200 or system 250 described herein.Referring to FIG. 12, shown is a diagrammatic illustration showingseveral cellularized filament grafts. Each cellularized filament graft320 includes the elongate cell growth substrate 321 and a population ofcells 322 attached to the substrate 321. The cells 322 can in certainembodiments form a generally confluent layer covering the cell growthsubstrate 321. To accomplish this, sufficient originally-provided cellscan be attached to the substrate 321 to form the layer, or the substratefilaments 320 can be seeded and then cultured sufficiently to form thegenerally confluent layer. In use, the cellularized filament grafts 320can be introduced individually into a site for treatment, for example bypositioning each strand cellularized filament graft 320 longitudinallywithin the lumen of a needle, inserting the needle into a desired targettissue, and driving the graft 320 from the needle with fluid pressure.The graft 320 will thereby distribute cells 322 through an elongateregion of the target tissue. This may be useful, for example, where thedevelopment of a vascular vessel or vessels in that region is desired.For these purposes, vessel-forming cells, such as endothelial cells orendothelial progenitor cells, including ECFC cells as discussed herein,can be used. In alternative embodiments, a flowable cell graftsuspension containing a plurality of elongate filament grafts 320 can beprovided within a syringe and injected into a target tissue. Such asuspension and a medical product for delivery thereof can be similar toproduct 300 depicted in FIG. 11, except using elongate grafts 320instead of, or in addition to, the relatively more compact particulatecellular graft bodies 303.

Referring now to FIG. 12A, shown is another cellularized filament graft320A of the invention. Graft 320A can have features which are the sameas graft 320 discussed above, including an elongate filament substrate321A and a population of cells 322A attached to the substrate. Graft320A further includes a patient tissue-engaging anchor element, which inthe illustrated embodiment is shown as a barb or hook 323A having a stem324A and a tissue engaging barb end 325A. The barb 323A or otheranchoring element in the illustrated embodiment is attached at orapproximate to an end of the filament substrate 321A. This attachmentcan be by tying, welding, bonding, integral formation with, or any othersuitable means. The anchoring element such as barb 323A in certainembodiments resists passage through tissue in one direction greater thanin an opposite direction. This can be achieved for example by thedirectional barb 325A. The barb 323A or other anchoring element can bemade of a persistent material or of a bioresorbable material.Illustratively, persistent or bioresorbable materials can be made frommetals or polymeric materials. Suitable bioresorbable polymers includepolymers of glycolic acid, polymers of lactic acid, or copolymers ofglycolic acid and lactic acid, polycaprolactone, and other knownmaterials. In use, the barb 323A or other anchoring element will resistmigration of the graft 320A once implanted in tissue of the patient,such as muscular or other tissue.

With reference now to FIG. 12B, in one mode of use, graft 320A can becombined with a delivery cannula 326A, such as a needle cannula. In oneform, the graft 320A can have all or a portion of the filament substrate320A received within a lumen 328A of the cannula 326A. Likewise, thebarb element 323A can be partially or wholly received within cannula326A, or, barb 323A can be resident completely out of cannula 326A, forinstance carried distally thereof. In the illustrative embodiment, stem324A or at least a portion thereof is received within the lumen in thedistal region of cannula 326A, and the barb end 325A is positionedbeyond the tissue-penetrated distal tip 327A of cannula 326A (e.g.needle tip). With the combined device in this condition the cannula 326Aand barb 325A can be used to penetrate into tissue of the patienttargeted for receipt of the graft 320A, by penetrating the patients skin“S” and driving the same to the desired depth in the underlying tissues.Thereafter, upon applying a withdrawing force to cannula 326A, the barbtip 325A will engage tissue of the patent and resist withdrawal whilethe cannula 326A is withdrawn thereby delivering the graft 320A from thelumen 328A as the cannula 326A is withdrawn. Graft 320A will then beleft implanted in the patent and cellular population 322A can in certainembodiments proliferate in the treatment of the patient. In someinventive variance, cellular population 322A includes endothelial cellsand/or endothelial progenitor cells, including for example endothelialcolony forming cells as discussed herein. The tissue to be treated andin which graft 320A is implanted can be tissue in need of vasculardevelopment, for example ischemic tissue of the myocardium or ischemictissue resultant of critical limb ischemia. In such uses, cellularpopulation 322A will be implanted positioned in an elongate region alongsubstrate 321A and can generate a vessel or vessels along the elongateimplant region. It will be understood, however, that other types ofcellular population 322A and other diseases, defects, or conditions canbe treated using filament graft 320A.

Cellular Grafts with Stacked Substrate Layers

Cellular grafts of the invention can also include multiple cell growthsubstrate sheets in a stacked configuration, with cellular populationsinterposed between the stacked sheets, and potentially also providingoutermost layers of the construct. To prepare such grafts, a first cellgrowth substrate layer can be seeded with cells, for example by applyinga liquid film containing a cell suspension to at least one side of thesheet. A second substrate layer can then be stacked onto the firstsubstrate layer against the side that had received the liquid film,followed by applying another cellular liquid film to the exposed side ofthe second substrate layer. This process can be repeated if desired toprovide a stacked substrate construct, with cells distributed evenly orregionally through the thickness of the construct. FIG. 13 provides aschematic diagram of one such stacked graft construct 330. Construct 330includes a plurality of stacked cell growth substrate layers 331,desirably harvested, purified extracellular matrix tissue sheets asdescribed herein. The stacked layers 331 can be partially or completelyoverlapped with one another. Cellular layers 332 each comprised of apopulation of cells 333 are provided between sheets 331, and optionallyalso to the outermost surfaces of construct 330. Prior to implantation,construct 330 can be incubated for a period of time at least sufficientfor cellular attachment to sheets 331, which can contribute to thestability of the construct as an integral graft unit. In onealternative, the construct 330 is incubated during a culture period toexpand the originally-seeded population of cells. Also, prior toapplying cells 333 to the sheets 331 during fabrication of the construct330, the sheets 331 can be preconditioned with blood components such asserum or serum protein(s) and/or with other culture media components,e.g. nutrients, salts, etc.

Stacked cellular graft constructs such as construct 330 can be preparedin automated systems such as system 200 of FIG. 9. To do so, a firstsubstrate sheet 331 can be provided in chamber 222, and applicationdevice 221 can be used to apply a processed cell suspension to the uppersurface of the sheet 331. A second substrate sheet 331 can then beoverlaid onto the first sheet, and application device 221 used to applyadditional amounts of the cell suspension to the second sheet 331. Thisprocess can be repeated multiple times, for example, two, three, four orfive times. Additionally, preconditioning media can be supplied frommedia unit 226 and applied to the respective sheets 331 prior to theapplication of the cell suspension. The first, second, and subsequentsheets can be sequentially positioned within chamber 222 manually by auser or using an automatic feed mechanism provided by system 200.

FIG. 17 shows another embodiment of a cellular graft of the inventionhaving stacked growth substrate sheets. Cellular graft 380 includes afirst cell growth substrate sheet 381, desirably any of theextracellular matrix layers identified herein, and a cellular material382 deposited on a surface of sheet 381. Cellular material 382 caninclude any of the cells identified herein along with a flowable cellgrowth material or substrate for example a particulate cell growthsubstrate as described herein and/or a gel cell growth substratematerial as described herein. After deposition of the material 382 onthe surface of sheet 381, a second cell growth substrate sheet 383 islayered over material 382 to create a stacked or sandwiched cellulargraft.

FIG. 18 illustrates still another embodiment of a cellular graft of theinvention. Graft 390 includes a first cell growth substrate sheet 391having a plurality of wells 392 defined therein and extending onlypartially through the thickness of sheet 391. A cellular material 393 isdeposited within wells 392. Cellular material 393 can for example besimply a cell population or can be cells combined with a flowable cellgrowth material such as a particulate substrate or gel-formed substrateor combination thereof, as described further herein. A second cellgrowth substrate sheet 394 is layered over the first sheet 391 so as tocover the filled wells 392 and optionally at least temporarily entrapcellular material 391 within wells 392. Cell growth substrate sheet 391and/or 394 and certain embodiments are any of the ECM layers asdescribed herein.

Cell Growth Substrate Articles with Flow-Directing Layers

The invention also provides articles of manufacture that include a cellgrowth substrate material covered or encapsulated within a secondmaterial that is less permeable to fluids such as aqueous cellularcompositions than the cell growth substrate material, wherein thecovering or encapsulating material can facilitate directing flow of acellular fluid through a length or thickness of the cell growthsubstrate material to aid in distributing the cells throughout thematerial. Referring to FIG. 14, shown is a cell growth substrate article340 that includes a cell growth substrate material 341 such as anydescribed herein, and a permeable or semi-permeable encapsulatingmaterial 342 enclosing the cell substrate material 341. Theencapsulating material 342 can define a first opening 343 and a secondopening 344 spaced from the first opening 343. In certain embodiments,the openings 343 and 344 will occur on opposed sides of the cell growthsubstrate material 341. Further, a plurality of openings 343 and/or 344could be provided in alternative embodiments. In use, article 340 can beconnected to a source of a liquid cellular suspension and the suspensionpassed into opening 343 under pressure, to thereby drive the cellularsuspension material through the cell growth substrate 341 to cause cellsto adhere or become lodged within the substrate 341. The fluid of thecell suspension can exit via opening 344, depopulated of at least someof the original cells. Optionally, fluid exiting opening 344 can berecirculated back through opening 343 to seed at least some remainingcells within substrate 341. The cell growth substrate material 341 canbe any monolithic, particulate, or other cell growth substrate materialdescribed herein. The encapsulating material 342 can be implantablewithin the patient, or can be a material not intended for implant whichcan be removed prior to implant of the cell growth substrate 341 afterseeding with cells. Encapsulating material 342 can for example be anatural or synthetic polymeric material, which can be persistent orbioresorbable upon implantation. Bioresorbable synthetic polymers, suchas those disclosed elsewhere herein, can be used for encapsulatingmaterial 342.

Referring to FIG. 15, shown is another cell growth substrate article ofthe invention. Article 350 includes a cell growth substrate 351 such asany of those disclosed herein, and a first encapsulating material 352 inwhich material 351 is received. Encapsulating material 352 is lesspermeable to a liquid in which cells are suspended or to be suspended,for example water or another aqueous medium, than the substrate material351. Encapsulating material 352 can for example be a plastic or otherpolymeric tray in which material 351 is received. The tray or otherencapsulating material 352 can define a first opening 353 and a secondopening 354 spaced from the first opening 353. A second encapsulatingmaterial 355 covers and seals an opening defined by tray or othermaterial 352. Encapsulating material 355 can for example be a polymericfilm peelably removable from tray or other encapsulating material 352.For these purposes, film or other material 355 can include an exposedand grippable portion 356, desirably at a periphery thereof, which canbe gripped and used to peel material 355 away from tray or othermaterial 352 to open the opening defined by tray or other material 352and expose the cell growth substrate 351 for removal. In this manner,during a cell-seeding operation such as that discussed above inconjunction with FIG. 14, material 355 will maintain a seal againstmaterial 352 and thus enclose cell growth substrate 351 to help guidefluid from opening 353 to opening 354 to seed cells through the volumeof material 351. After the seeding process, the encapsulating material355 can be peeled from the material 352 and the seeded substrate 351removed for implant into the patient.

Referring now to FIG. 16, shown is another embodiment of a cell growthsubstrate article 360. Article 360 is similar in many respects toarticle 350 discussed above, and thus has features which arecorrespondingly numbered except in the “360” series. Article 360 furtherincludes fluid distribution features associated with at least the fluidentry opening 363 and preferably with both that opening and fluid exitopening 364. Desirably, these features facilitate a substantially plugflow of liquid through article 360, with the plug having a crosssectional dimension substantially in the shape of the cross section ofsubstrate 361. This will help to evenly distribute the cells through thesubstrate 361. For these purposes, tray or other encapsulating material362 defines diverting walls 367 and 368 which diverge as they travelaway from opening 363 and toward the periphery of cell growth substrate361. Desirably, the inner surfaces of diverging walls 367 and 368diverge two points substantially at the outer periphery of cell growthsubstrate 361. In this fashion, fluids entering opening 363 will fillthe defined void 369 proximal of substrate 361, whereafter pressure offluid entering opening 363 will be evenly distributed and cause asubstantial plug flow through the substrate 361 across its crosssectional dimension. Fluids traversing substrate 361 will then becollected in void 373 defined by walls 371 and 372 which converge in thedirection of exit opening 364. These and other features for facilitatinga substantial plug flow across the substrate 361 within article 360 arecontemplated in accordance with the invention.

Cell Growth Substrate Materials for Use in Inventive Embodiments

As noted above, cell growth substrates of and used in the invention cancomprise extracellular matrix (ECM) tissue. The ECM tissue can beobtained from a warm-blooded vertebrate animal, such as an ovine, bovineor porcine animal. For example, suitable ECM tissue include thosecomprising submucosa, renal capsule membrane, dermal collagen, duramater, pericardium, fascia lata, serosa, peritoneum or basement membranelayers, including liver basement membrane. Suitable submucosa materialsfor these purposes include, for instance, intestinal submucosa includingsmall intestinal submucosa, stomach submucosa, urinary bladdersubmucosa, and uterine submucosa. ECM tissues comprising submucosa(potentially along with other associated tissues) useful in the presentinvention can be obtained by harvesting such tissue sources anddelaminating the submucosa-containing matrix from smooth muscle layers,mucosal layers, and/or other layers occurring in the tissue source.Porcine tissue sources are preferred sources from which to harvest ECMtissues, including submucosa-containing ECM tissues.

ECM tissue when used in the invention is preferably decellularized andhighly purified, for example, as described in U.S. Pat. No. 6,206,931 toCook et al. or U.S. Patent Application Publication No. US2008286268dated Nov. 20, 2008, publishing U.S. patent application Ser. No.12/178,321 filed Jul. 23, 2008, all of which are hereby incorporatedherein by reference in their entirety. Preferred ECM tissue materialwill exhibit an endotoxin level of less than about 12 endotoxin units(EU) per gram, more preferably less than about 5 EU per gram, and mostpreferably less than about 1 EU per gram. As additional preferences, thesubmucosa or other ECM material may have a bioburden of less than about1 colony forming units (CFU) per gram, more preferably less than about0.5 CFU per gram. Fungus levels are desirably similarly low, for exampleless than about 1 CFU per gram, more preferably less than about 0.5 CFUper gram. Nucleic acid levels are preferably less than about 5 μg/mg,more preferably less than about 2 μg/mg, and virus levels are preferablyless than about 50 plaque forming units (PFU) per gram, more preferablyless than about 5 PFU per gram. These and additional properties ofsubmucosa or other ECM tissue taught in U.S. Pat. No. 6,206,931 or U.S.Patent Application Publication No. US2008286268 may be characteristic ofany ECM tissue used in the present invention.

In certain embodiments, the ECM tissue material used as or in the cellgrowth substrate will be a membranous tissue with a sheet structure asisolated from the tissue source. The ECM tissue can, as isolated, have alayer thickness that ranges from about 50 to about 250 microns whenfully hydrated, more typically from about 50 to about 200 microns whenfully hydrated, although isolated layers having other thicknesses mayalso be obtained and used. These layer thicknesses may vary with thetype and age of the animal used as the tissue source. As well, theselayer thicknesses may vary with the source of the tissue obtained fromthe animal source.

The ECM tissue material utilized desirably retains a structuralmicroarchitecture from the source tissue, including structural fiberproteins such as collagen and/or elastin that are non-randomly oriented.Such non-random collagen and/or other structural protein fibers can incertain embodiments provide an ECM tissue that is non-isotropic inregard to tensile strength, thus having a tensile strength in onedirection that differs from the tensile strength in at least one otherdirection.

The ECM tissue material may include one or more bioactive agents nativeto the source of the ECM tissue material and retained in the ECM tissuematerial through processing. For example, a submucosa or otherremodelable ECM tissue material may retain one or more native growthfactors such as but not limited to basic fibroblast growth factor(FGF-2), transforming growth factor beta (TGF-beta), epidermal growthfactor (EGF), cartilage derived growth factor (CDGF), and/or plateletderived growth factor (PDGF). As well, submucosa or other ECM materialswhen used in the invention may retain other native bioactive agents suchas but not limited to proteins, glycoproteins, proteoglycans, andglycosaminoglycans. For example, ECM materials may include heparin,heparin sulfate, hyaluronic acid, fibronectin, cytokines, and the like.Thus, generally speaking, a submucosa or other ECM material may retainfrom the source tissue one or more bioactive components that induce,directly or indirectly, a cellular response such as a change in cellmorphology, proliferation, growth, protein or gene expression.

Submucosa-containing or other ECM materials used in the presentinvention can be derived from any suitable organ or other tissue source,usually sources containing connective tissues. The ECM materialsprocessed for use in the invention will typically include abundantcollagen, most commonly being constituted at least about 80% by weightcollagen on a dry weight basis. Such naturally-derived ECM materialswill for the most part include collagen fibers that are non-randomlyoriented, for instance occurring as generally uniaxial or multi-axialbut regularly oriented fibers. When processed to retain native bioactivefactors, the ECM material can retain these factors interspersed assolids between, upon and/or within the collagen fibers. Particularlydesirable naturally-derived ECM materials for use in the invention willinclude significant amounts of such interspersed, non-collagenous solidsthat are readily ascertainable under light microscopic examination withappropriate staining. Such non-collagenous solids can constitute asignificant percentage of the dry weight of the ECM material in certaininventive embodiments, for example at least about 1%, at least about 3%,and at least about 5% by weight in various embodiments of the invention.

The submucosa-containing or other ECM material used in the presentinvention may also exhibit an angiogenic character and thus be effectiveto induce angiogenesis in a host engrafted with the material. In thisregard, angiogenesis is the process through which the body makes newblood vessels to generate increased blood supply to tissues. Thus,angiogenic materials, when contacted with host tissues, promote orencourage the formation of new blood vessels into the materials. Methodsfor measuring in vivo angiogenesis in response to biomaterialimplantation have recently been developed. For example, one such methoduses a subcutaneous implant model to determine the angiogenic characterof a material. See, C. Heeschen et al., Nature Medicine 7 (2001), No. 7,833-839. When combined with a fluorescence microangiography technique,this model can provide both quantitative and qualitative measures ofangiogenesis into biomaterials. C. Johnson et al., Circulation Research94 (2004), No. 2, 262-268.

Further, in addition or as an alternative to the inclusion of suchnative bioactive components, non-native bioactive components such asthose synthetically produced by recombinant technology or other methods(e.g., genetic material such as DNA), may be incorporated into an ECMmaterial used in the invention. These non-native bioactive componentsmay be naturally-derived or recombinantly produced proteins thatcorrespond to those natively occurring in an ECM tissue, but perhaps ofa different species. These non-native bioactive components may also bedrug substances. Illustrative drug substances that may be added tomaterials include, for example, anti-clotting agents, e.g. heparin,antibiotics, anti-inflammatory agents, thrombus-promoting substancessuch as blood clotting factors, e.g., thrombin, fibrinogen, and thelike, and anti-proliferative agents, e.g. taxol derivatives such aspaclitaxel. Such non-native bioactive components can be incorporatedinto and/or onto ECM material in any suitable manner, for example, bysurface treatment (e.g., spraying) and/or impregnation (e.g., soaking),just to name a few. Also, these substances may be applied to the ECMmaterial in a premanufacturing step, immediately prior to the procedure(e.g., by soaking the material in a solution containing a suitableantibiotic such as cefazolin), or during or after engraftment of thematerial in the patient.

Inventive graft compositions herein can incorporate xenograft ECMmaterial (i.e., cross-species material, such as tissue material from anon-human donor to a human recipient), allograft ECM material (i.e.,interspecies material, with tissue material from a donor of the samespecies as the recipient), and/or autograft ECM material (i.e., wherethe donor and the recipient are the same individual). Further, anyexogenous bioactive substances incorporated into an ECM material may befrom the same species of animal from which the ECM material was derived(e.g. autologous or allogenic relative to the ECM material) or may befrom a different species from the ECM material source (xenogenicrelative to the ECM material). In certain embodiments, ECM tissuematerial will be xenogenic relative to the patient receiving the graft,and any added cells or other exogenous material(s) will be from the samespecies (e.g. autologous or allogenic) as the patient receiving thegraft. Illustratively, human patients may be treated with xenogenic ECMmaterials (e.g. porcine-, bovine- or ovine-derived) that have beenmodified with exogenous human cells and/or serum proteins and/or othermaterial(s) as described herein, those exogenous materials beingnaturally derived and/or recombinantly produced.

When used in the invention, ECM materials can be free or essentiallyfree of additional, non-native crosslinking, or may contain additionalcrosslinking. Such additional crosslinking may be achieved byphoto-crosslinking techniques, by chemical crosslinkers, or by proteincrosslinking induced by dehydration or other means. However, becausecertain crosslinking techniques, certain crosslinking agents, and/orcertain degrees of crosslinking can destroy the remodelable propertiesof a remodelable material, where preservation of remodelable propertiesis desired, any crosslinking of the remodelable ECM material can beperformed to an extent or in a fashion that allows the material toretain at least a portion of its remodelable properties. Chemicalcrosslinkers that may be used include for example aldehydes such asglutaraldehydes, diimides such as carbodiimides, e.g.,1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, ribose orother sugars, acyl-azide, sulfo-N-hydroxysuccinamide, or polyepoxidecompounds, including for example polyglycidyl ethers such asethyleneglycol diglycidyl ether, available under the trade name DENACOLEX810 from Nagese Chemical Co., Osaka, Japan, and glycerol polyglycerolether available under the trade name DENACOL EX 313 also from NageseChemical Co. Typically, when used, polyglycerol ethers or otherpolyepoxide compounds will have from 2 to about 10 epoxide groups permolecule.

In additional embodiments, substrates of the invention can be made fromECM's or other collagenous materials that have been subjected toprocesses that expand the materials. In certain forms, such expandedmaterials can be formed by the controlled contact of an ECM materialwith a denaturing agent such as one or more alkaline substances untilthe material expands, and the isolation of the expanded material.Illustratively, the contacting can be sufficient to expand the ECMmaterial to at least 120% of (i.e. 1.2 times) its original bulk volume,or in some forms to at least about two times its original volume.Thereafter, the expanded material can optionally be isolated from thealkaline medium, e.g. by neutralization and/or rinsing. The collected,expanded material can be used in any suitable manner in the preparationof a substrate. Illustratively, the expanded material can be enrichedwith bioactive components, comminuted, dried, and/or molded, etc., inthe formation of a substrate of a desired shape or configuration. Incertain embodiments, a dried substrate formed with the expanded ECMmaterial can be highly compressible and/or expandable.

Treatment of an ECM material with a denaturant, such as an alkalinematerial, can cause changes in the physical structure of the materialthat in turn cause it to expand. Such changes may include denaturationof the collagen in the material. In certain embodiments, it is preferredto expand the material to at least about three, at least about four, atleast about 5, or at least about 6 or even more times its original bulkvolume. It will be apparent to one skilled in the art that the magnitudeof the expansion is related to several factors, including for instancethe concentration or pH of the alkaline medium, the exposure time of thealkaline medium to the material, and temperature used in the treatmentof the material to be expanded, among others. These factors can bevaried through routine experimentation to achieve a material having thedesired level of expansion, given the disclosures herein.

A collagen fibril is comprised of a quarter-staggered array oftropocollagen molecules. The tropocollagen molecules themselves areformed from three polypeptide chains linked together by covalentintramolecular bonds and hydrogen bonds to form a triple helix.Additionally, covalent intermolecular bonds are formed between differenttropocollagen molecules within the collagen fibril. Frequently, multiplecollagen fibrils assemble with one another to form collagen fibers. Itis believed that the addition of an alkaline substance to the materialas described herein can be conducted so as to not significantly disruptthe intramolecular and intermolecular bonds, but denature the materialto an extent that provides to the material an increased processedthickness, e.g. at least twice the naturally-occurring thickness. ECMmaterials that can be processed to make expanded materials for use assubstrates can include any of those disclosed herein or other suitableECM's. Typical such ECM materials will include a network of collagenfibrils having naturally-occurring intramolecular cross links andnaturally-occurring intermolecular cross links. Upon expansionprocessing as described herein, the naturally-occurring intramolecularcross links and naturally-occurring intermolecular cross links can beretained in the processed collagenous matrix material sufficiently tomaintain the collagenous matrix material as an intact collagenous sheetmaterial; however, collagen fibrils in the collagenous sheet materialcan be denatured, and the collagenous sheet material can have analkaline-processed thickness that is greater than the thickness of thestarting material, for example at least 120% of the original thickness,or at least twice the original thickness. The expanded ECM material canthen be processed to provide foam or sponge substrates, e.g. bycomminuting, casting, and drying the processed material. Additionalinformation concerning expanded ECM materials and their preparation isfound in United States Patent Application Publication No. US20090326577published Dec. 31, 2009, publishing U.S. patent application Ser. No.12/489,199 filed Jun. 22, 2009, which is hereby incorporated herein byreference in its entirety.

In addition to or as an alternative to ECM materials, the cell growthsubstrate used in the invention may be comprised of other suitablematerials. Illustrative materials include, for example,synthetically-produced substrates comprised or natural or syntheticpolymers. Illustrative synthetic polymers can include nonresorbablesynthetic biocompatible polymers, such as cellulose acetate, cellulosenitrate, silicone, polyethylene teraphthalate, polyurethane, polyamide,polyester, polyorthoester, polyanhydride, polyether sulfone,polycarbonate, polypropylene, high molecular weight polyethylene,polytetrafluoroethylene, or mixtures or copolymers thereof; orresorbable synthetic polymer materials such as polylactic acid,polyglycolic acid or copolymers thereof, polyanhydride,polycaprolactone, polyhydroxy-butyrate valerate, polyhydroxyalkanoate,or another biodegradable polymer or mixture thereof. Preferred cellgrowth substrates comprised of these or other materials will be porousmatrix materials configured to allow cellular invasion and ingrowth intothe matrix.

Cells for Use in Inventive Embodiments

Any one or any combination of a wide variety of cell types can be usedin cellular graft-related compositions and methods of the invention. Forexample, the cells can be skin cells, skeletal muscle cells, cardiacmuscle cells, lung cells, mesentery cells, or adipose cells. The adiposecells may be from omental fat, properitoneal fat, perirenal fat,pericardial fat, subcutaneous fat, breast fat, or epididymal fat. Incertain embodiments, the cells comprise stromal cells, stem cells, orcombinations thereof. As used herein, the term “stem cells” is used in abroad sense and includes traditional stem cells, adipose derived stemcells, progenitor cells, preprogenitor cells, reserve cells, and thelike. Exemplary stem cells include embryonic stem cells, adult stemcells, pluripotent stem cells, neural stem cells, liver stem cells,muscle stem cells, muscle precursor stem cells, endothelial progenitorcells, bone marrow stem cells, chondrogenic stem cells, lymphoid stemcells, mesenchymal stem cells, hematopoietic stem cells, central nervoussystem stem cells, peripheral nervous system stem cells, and the like.Additional illustrative cells which can be used include hepatocytes,epithelial cells, Kupffer cells, fibroblasts, neurons, cardiomyocytes,myocytes, chondrocytes, pancreatic acinar cells, islets of Langerhans,osteocytes, myoblasts, satellite cells, endothelial cells, adipocytes,preadipocytes, biliary epithelial cells, and progentior cells of any ofthese cell types.

In some embodiments, the cells incorporated in the cellular grafts are,or include, endothelial progenitor cells (EPCs). Preferred EPCs for usein the invention are endothelial colony forming cells (ECFCs),especially ECFCs with high proliferative potential. Suitable such cellsare described for example in U.S. Patent Application Publication No.20050266556 published Dec. 1, 2005, publishing U.S. patent applicationSer. No. 11/055,182 filed Feb. 9, 2005, and U.S. Patent ApplicationPublication No. 20080025956 published Jan. 1, 2008, publishing U.S.patent application Ser. No. 11/837,999, filed Aug. 13, 2007, each ofwhich is hereby incorporated by reference in its entirety. Such ECFCcells can be a clonal population, and/or can be obtained from umbilicalcord blood of humans or other animals. Additionally or alternatively,the endothelial colony forming cells have the following characteristics:(a) express the cell surface antigens CD31, CD105, CD146, and CD144;and/or (b) do not express CD45 and CD14; and/or (c) ingest acetylatedLDL; and/or (d) replate into at least secondary colonies of at least2000 cells when plated from a single cell; and/or (e) express highlevels of telomerase, at least 34% of that expressed by HeLa cells;and/or (f) exhibit a nuclear to cytoplasmic ratio that is greater than0.8; and/or (g) have cell diameters of less than about 22 microns. Anycombination of some or all of these features (a)-(g) may characterizeECFCs used in the present invention.

In other embodiments, the cells incorporated in the cellular grafts are,or include, muscle derived cells, including muscle derived myoblastsand/or muscle derived stem cells. Suitable such stem cells and methodsfor obtaining them are described, for example, in U.S. Pat. No.6,866,842 and U.S. Pat. No. 7,155,417, each of which is herebyincorporated herein by reference in its entirety. The muscle derivedcells can express desmin, M-cadherin, MyoD, myogenin, CD34, and/orBcl-2, and can lack expression of CD45 or c-Kit cell markers.

In still other embodiments, the cells incorporated in the cellulargrafts are, or include, stem cells derived from adipose tissue. Suitablesuch cells and methods for obtaining them are described for example inU.S. Pat. No. 6,777,231 and U.S. Pat. No. 7,595,043, each of which ishereby incorporated herein by reference in its entirety. The cellularpopulation can include adipose-derived stem and regenerative cells,sometimes also referred to as stromal vascular fraction cells, which canbe a mixed population including stem cells, endothelial progenitorcells, leukocytes, endothelial cells, and vascular smooth muscle cells,which can be adult-derived. In certain forms, cellular grafts of thepresent invention can be prepared with and can include adipose-derivedcells that can differentiate into two or more of a bone cell, acartilage cell, a nerve cell, or a muscle cell.

Medical Treatments with Cellular Grafts

Cellular grafts of and prepared in accordance with the invention can beused in a wide variety of clinical applications to treat damaged,diseased or insufficient tissues, and can be used in humans or innon-human animals. Such tissues to be treated may, for example, bemuscle tissue, nerve tissue, brain tissue, blood, myocardial tissue,cartilage tissue, organ tissue such as lung, kidney or liver tissue,bone tissue, arterial or venous vessel tissue, skin tissue, and others.

In certain embodiments, the cellular grafts can be used to enhance theformation of blood vessels in a patient, for example to alleviateischemia in tissues. Direct administration of blood vessel-formingcellular grafts, for example grafts containing endothelial colonyforming cells or other endothelial progenitor cells, to an ischemic sitecan enhance the formation of new vessels in the affected areas andimprove blood flow or other outcomes. The ischemic tissue to be treatedmay for example be ischemic myocardial tissue, e.g. following aninfarction, or ischemic tissue in the legs or other limbs such as occursin critical limb ischemia. The cellular graft administered to theischemic tissue can be a flowable graft material, and in particular aninjectable graft material, as disclosed herein.

The cellular grafts can also be used to enhance the healing of partialor full thickness dermal wounds, such as skin ulcers, e.g. diabeticulcers, and burns. Illustratively, the administration of graftscontaining endothelial colony forming cells or other endothelialprogenitor cells to such wounds can enhance the healing of the wounds.

In other applications, the cellular grafts can be used to generatemuscle tissue at a target site, for example in the treatment of skeletalmuscle tissue, smooth muscle tissue, myocardial tissue, or other tissue.Illustratively, cellular grafts of the invention containing musclederived myoblasts can be delivered, e.g. by injection, into muscletissue of a sphincter such as a urinary bladder sphincter to treatincontinence.

It will be understood that all cell-containing graft materialembodiments as described herein, and method embodiments as describedherein, can be prepared and conducted using cell-seeding devices andsystems as described herein, for example including generally the stepsof loading the substrate material into the cell-seeding device, loadinga cellular composition into the cell-seeding device, and combining thesubstrate material and cells at least in part, and potentiallycompletely, through the operation of the cell-seeding device. When othercomponents described herein, such as a preconditioning medium, gel orgellable material, etc., are used, they can be charged to theappropriate chamber or passage of the cell-seeding device for operationand combination with the other material(s), as generally described,under the action of the cell-seeding device. Those skilled in the artwill readily understand these combinations of features and embodimentsdescribed herein, which constitute further aspects of the invention. Thecell-seeded graft material can then be obtained from the cell-seedingdevice, and optionally administered to a patient, including a humanpatient, e.g. for the medical indications identified herein.

The uses of the terms “a” and “an” and “the” and similar references inthe context of describing the invention (especially in the context ofthe following claims) are to be construed to cover both the singular andthe plural, unless otherwise indicated herein or clearly contradicted bycontext. Recitation of ranges of values herein are merely intended toserve as a shorthand method of referring individually to each separatevalue falling within the range, unless otherwise indicated herein, andeach separate value is incorporated into the specification as if it wereindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g., “such as”) provided herein, isintended merely to better illuminate the invention and does not pose alimitation on the scope of the invention unless otherwise claimed. Nolanguage in the specification should be construed as indicating anynon-claimed element as essential to the practice of the invention.

While the invention has been illustrated and described in detail in thedrawings and foregoing description, the same is to be considered asillustrative and not restrictive in character, it being understood thatonly the preferred embodiment has been shown and described and that allchanges and modifications that come within the spirit of the inventionare desired to be protected. In addition, all references cited hereinare indicative of the level of skill in the art and are herebyincorporated by reference in their entirety.

1-10. (canceled)
 11. A method for preparing a cell-seeded compositionfor delivery to a patient, comprising: providing a fluid extracellularmatrix particulate composition comprising particles of extracellularmatrix and a liquid medium; combining the fluid extracellular matrixcomposition with a liquid cell suspension to form a cellular fluidcomposition; and mixing the cellular fluid composition.
 12. The methodof claim 11, also comprising causing said cellular fluid composition togel.
 13. The method of claim 12, wherein said causing comprises alteringthe pH of the cellular fluid composition.
 14. The method of claim 12,wherein said causing comprises altering the temperature of the cellularfluid composition. 15-49. (canceled)
 50. A method for treating apatient, comprising: combining a suspension of cells with a particulatecell growth substrate; incubating the suspension of cells in contactwith the particulate cell growth substrate to form cellularizedparticulate bodies having cells attached to particles of the particulatecell growth substrate, wherein said incubating is for a duration andunder conditions such that significant expansion of the number of cellsdoes not occur; and administering said cellularized particulate bodiesto the patient.
 51. The method of claim 50, wherein said incubating isfor a duration and under conditions effective to achieve attachment ofat least 20% of the cells in said suspension to said particles butwithout expansion of the number of cells.
 52. A method for treating apatient, comprising: introducing a cellular graft into the patient,wherein the cellular graft includes cellularized particulate bodiescomprised of cells attached to extracellular matrix particles. 53-55.(canceled)
 56. A cellular graft material, comprising: a particulate cellgrowth substrate material, and cells attached to particles of said cellgrowth substrate material, wherein the cells comprise endothelialprogenitor cells, muscle derived cells, or a combination thereof. 57-61.(canceled)
 62. A cell growth substrate composition, comprising: aparticulate material comprising sheet-form cell growth substrateparticles have a compact shape.
 63. The substrate composition of claim62, wherein at least 25% of the sheet-form substrate particles, whenconsidered in the plane of the sheet, have a first, maximum crosssectional dimension axis which is no more than about two times thelength of a second cross sectional axis taken on a line perpendicular toand centered upon the maximum cross sectional dimension axis.
 64. Thecomposition of claim 62, wherein the substrate particles have a maximumcross sectional dimension in the range of about 20 microns to about 2000microns.
 65. An extracellular matrix composition, comprising: aparticulate extracellular matrix material comprising sheet-formextracellular matrix particles having a compact shape.
 66. Thecomposition of claim 65, wherein at least 25% of the sheet-formextracellular matrix particles, when considered in the plane of thesheet, have a first, maximum cross sectional dimension axis which is nomore than about two times the length of a second cross sectional axistaken on a line perpendicular to and centered upon the maximum crosssectional dimension axis.
 67. The composition of claim 65, wherein theextracellular matrix particles have a maximum cross sectional dimensionin the range of about 20 microns to about 2000 microns.
 68. Thecomposition of claim 66, wherein the extracellular matrix particles havea maximum cross sectional dimension in the range of about 20 microns toabout 2000 microns.
 69. The composition of claim 62, also comprisingcells attached to the particles.
 70. The composition of claim 69,wherein the cells cover substantially the entire outer surface of theparticles.
 71. The composition of claim 70, wherein the cells form asubstantially confluent monolayer covering the entire outer surface ofthe particles.
 72. The composition of claim 71, wherein the cellscomprise endothelial cells, endothelial progenitor cells, or a mixturethereof.
 73. The composition of claim 72, wherein the cells are clonal.74. The composition of claim 72, wherein the cells consist ofendothelial cells, endothelial progenitor cells, or a mixture thereof.75-87. (canceled)